Proc. Nail. Acad. Sci. USA Vol. 83, pp. 4962-4966, July 1986 Neurobiology Identification of three forms of human basic by cDNA cloning (myelin /oligodendroglia/) JOHN KAMHOLZ*, FRANCESCA DE FERRAt, CARMIE PUCKETT*, AND ROBERT LAZZARINI* *Laboratory of Molecular , National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892; and tThe Wistar Institute, 36th Street and Spruce, Philadelphia, PA 19104 Communicated by Roscoe 0. Brady, March 10, 1986

ABSTRACT We have isolated cDNA clones encoding three Unlike the case in the mouse, only a single MBP species separate forms of human myelin basic protein (MBP), 21.5, has been identified in human myelin. The human 18.5-kDa 18.5, and 17.2 kDa, and have determined the nucleotide MBP has been well characterized, and its amino acid se- sequence of each. The three forms share a common sequence quence has been determined directly (6). Although minor but differ by the inclusion of a 26-residue amino acid sequence bands, both larger (7) and smaller (8), are sometimes seen on near the N terminus of the 21.5-kDa protein or by the absence protein gels of human MBP, these forms have not been ofan 11-residue amino acid sequence near the C terminus ofthe isolated or characterized. 17.2-kDa protein. The sequences either added to or deleted We have examined the question ofmultiple forms ofhuman from the major 18.5-kDa MBP correspond exactly to exons 2 MBP by assembling a large collection of human MBP cDNA and 5 of the mouse MBP , suggesting that the human and clones and examining their structure by restriction endonu- mouse have similar exon structures. We have also clease mapping and nucleotide . We have identi- identified the 21.5-kDa human MBP on immunoblots using fied three types of MBP cDNAs. The most common human antisera raised to a peptide encoded by the mouse exon 2 MBP cDNA corresponds to the 18.5-kDa MBP; the amino sequence. Southern blotting studies of human genomic DNA acid sequence predicted from its nucleotide sequence match- reveal a simple pattern consistent with a single human MBP es precisely that reported for the 18.5 MBP. The two other gene. Thus, the three MBP mRNAs are likely to arise from types of MBP cDNAs encode two previously undescribed alternative splicing of a primary human MBP transcript. isoforms ofhuman MBP, one of21.5 kDa and a second of 17.2 Conservation of the 26 amino acid mouse exon 2 sequence in kDa. The 21.5-kDa human protein, like its equivalent in human MBP suggests an important role for this sequence in mouse, contains an extra 26 amino acid sequence near its N myelination. terminus. The 17.2-kDa protein is identical to the human 18.5 kDa protein but is missing 11 amino acids near its C terminus. Myelin is a multilamellar compacted membrane structure that Comparison of human and mouse MBP isoforms suggests surrounds and electrically insulates the , facilitating the that the human and mouse MBP genes have similar structures conduction of impulses. This elaborate structure is and that the human MBPs also arise from alternative splicing synthesized and assembled by in the cen- of a primary human MBP transcript. tral (CNS) and Schwann cells in the periph- eral nervous system (PNS) (1). Myelin basic protein (MBP) MATERIALS AND METHODS constitutes 30% ofthe total myelin protein in the CNS, but it is a lesser constituent of PNS myelin. The function of MBP Preparation of RNA and DNA. Total RNA was prepared is not known, but its location on the cytoplasmic side of the from by the guanidine thiocyanate/cesium myelin membrane and its tendency to self-aggregate suggest chloride method (9). Fractions enriched in mRNA were that MBP stabilizes the myelin membrane by linking the prepared from total RNA by oligo(dT) selection (10). RNA apposed cytoplasmic surfaces of the membrane sheaths. was fractionated for blot hybridization in agarose/formalde- Myelination occurs during a relatively brief time period hyde gels (10). High molecular weight DNA was prepared postnatally, and a characteristic temporal sequence for the from human placenta. synthesis of MBP prior to the onset of myelination has been Construction of Human Brain cDNA Libraries. Human described (2, 3). brain autopsy material was obtained (through the courtesy of Mouse CNS myelin contains four structurally related Lucy Rorke, Children's Hospital of Philadelphia) from a forms of MBP with molecular masses of21.5, 18.5, 17.0, and 1-day female infant who succumbed to congenital heart 14.0 kDa. We (4) and Takahashi et al. (5) have recently disease and (through the courtesy of John Sever of the determined that these four forms of mouse MBP are pro- Infectious Diseases Branch, National Institute of Neurolog- duced by alternative splicing of the primary transcript from ical and Communicative Disorders and Stroke) from a patient a single MBP gene. The gene consists of seven exons, two of with subacute sclerosing panencephalitis (SSPE). mRNA which, exons 2 and 6, are alternatively spliced, accounting was prepared from the spinal cord, the basal ganglia, and a for all four forms of mouse MBP. The largest form of MBP section ofbrain stem/cerebellum at the level of the mid pons contains the amino acid sequences encoded by all seven from the infant material and from the cerebellum of the exons. The 18.5-kDa and the 17.0-kDa proteins are homol- patient with SSPE. Expression libraries were constructed in ogous to the largest form but are missing the portions of the the vector Xgtll essentially as described by Huynh et al. (11). sequence encoded by exon 6 and exon 2, respectively. The An oligo(dT) primer was used to reverse transcribe the smallest form of MBP (14.0 kDa) lacks the sequences mRNA. The double-stranded cDNAs were ligated to EcoRI encoded by both exons 2 and 6. linkers and sized by acrylamide gel electrophoresis. The fraction identified by autoradiography as larger than 800 base The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: MBP, myelin basic protein; ORF, open reading in accordance with 18 U.S.C. §1734 solely to indicate this fact. frame; kb, kilobase(s); bp, (s). 4962 Downloaded by guest on September 26, 2021 Neurobiology: Kamholz et al. Proc. Natl. Acad. Sci. USA 83 (1986) 4963 pairs (bp) was recovered from the gel by elution in 20 mM Coding Region Probe Non-Coding Region Probe Tris HCl, pH 7.4/20 mM NaCl/1 mM EDTA for 24 hr, ligated Human Mouse Human Mouse to Xgt1l arms, and packaged in vitro into phage particles. The M H rRNA M H rRNA M HrRNA number of independent clones contained in each library was M H rRNA as follows: basal ganglia, 1,000,000; brain stem/cerebellum, I I t .. 4,000,000; spinal cord, 175,000; SSPE, 310,000. Partial char- I. acterization of these libraries has been previously reported 1. (12). Plaques of phages bearing MBP sequences were identified after transfer to nylon membranes by hybridization to a nick-translated insert from pdFl91, a mouse cDNA clone that -.1 contains most of the MBP coding region. The filters were hybridized in 5x SSPE (lx SSPE is 0.15 M NaCl/0.015 M 0 5x Denhardt's rw monosodium phosphate/i mM Na2EDTA), solution (lx Denhardt's solution is 0.02% Ficoll/0.02% polyvinylpyrrolidone/0.02% bovine serum albumin), 1% NaDodSO4, and 40% (vol/vol) formamide containing soni- cated salmon sperm DNA at 100 jug/ml at 420C for 16 hr, FIG. 1. Blot hybridization analysis ofhuman and mouse poly(A)+ RNA. Hybridization was carried out as described in Materials and followed by two washes in 2x SSC (lx SSC is 0.15 M Methods for library screening except that the formamide concentra- NaCl/0.015 M sodium citrate)/0.1% NaDodSO4 at room tion was 50%6 (vol/vol). The amount loaded per lane was 2 ,ug of temperature and a final wash in 2x SSC/0.1% NaDodSO4 at poly(A)+ RNA from 18-day mouse brain (M) or 4 ,ug of poly(A)+ 570C. RNA from human brain (H). The right lanes contained '4C-labeled DNA Sequence Analysis. DNA sequencing was carried out 18S and 28S rRNA markers. by the dideoxy chain termination (13) and the chemical cleavage (14) methods. foreshadowed in Fig. 1, the untranslated regions were less Immunoblotting. Immunoblotting was carried out as de- homologous (70%). The 800-bp open reading frame (ORF) in scribed by Towbin et al. (15). Peptides were synthesized and the 3' untranslated portion ofthe rat and mouse cDNAs is not were prepared as previously described (16). present in the human, although there is a 354-bp ORF General Methods. Unless otherwise stated, all methods (nucleotides 784-1137) in the human 3' sequence that over- were as described by Maniatis et al. (10). All radioactive laps a portion of the mouse ORF. Unlike the situation in the probes were prepared by nick-translation of purified DNA mouse and rat mRNAs, the second ORF of the human MBP inserts. does not contain in-frame consensus splice acceptor sites. The human and mouse ORFs share a putative 14 amino acid sequence (underlined in the figure) but are otherwise non- RESULTS homologous. The amino acid sequence deduced from the Isolation of a Human 18.5-kDa MBP cDNA. The human human MBP cDNA agrees entirely with the amino acid brain stem/cerebellum cDNA library was screened at re- sequence of the 18.5-kDa human MBP as determined by duced stringency for MBP sequences, using a mouse MBP Carnegie (6) with corrections by Gibson et al. (17). cDNA clone, pdFl91, as a hybridization probe. This plasmid Human MBP Gene. A Southern blot of human placental contains the entire coding region ofthe 14.0-kDa mouse MBP DNA probed with a nick-translated insert from either pHBP- except for the last seven amino acids and detects both a 1 or pHBP-2 is shown in Fig. 3. The fragment patterns 2.1-kilobase (kb) mouse mRNA and a 2.2-kb human mRNA produced are relatively simple and suggest that the human, in blot hybridizations (data not shown). We screened 250,000 like the mouse, has a single copy ofthe MBP gene per haploid independent plaques and identified 50 positive clones. Sev- genome. eral ofthese clones contained inserts of2.2 kb and were, thus, Identification ofa 21.5-kDa Human MBP. Although a major near full-length copies of human MBP mRNA. The insert of 18.5-kDa MBP has been characterized from human myelin, one ofthese clones was cleaved by EcoRP into two fragments, several authors have suggested there may be minor forms of 1.2 and 1.0 kb, the larger of which was shown by Southern the protein, both larger and smaller. Sheep (18), cattle (19), hybridization, using pdFl91 as a probe, to contain the MBP and rabbits (20) have been shown to have small amounts of coding region sequences. These two EcoRI fragments were a putative 21.5-kDa MBP as well as the 18.5-kDa form. If subcloned in pUC8 and designated pHBP-1 (1.2-kb coding there were multiple forms of human MBP, some of them fragment) and pHBP-2 (1.0-kb noncoding fragment). might contain sequences homologous to those encoded by Blot hybridization analysis of human and mouse brain mouse exon 2, which is found only in the 21.5- and 17.0-kDa poly(A)+-RNA probed with either coding or noncoding forms of mouse MBP. To test this hypothesis, a 72-bp Rsa I sequences from mouse and human MBP cDNAs is shown in fragment containing only mouse exon 2 sequences was Fig. 1. Both coding region probes detect mouse and human isolated from a mouse cDNA clone and used as a probe in mRNA. However, the noncoding probes are species specific both Southern transfer hybridization ofhuman genomic DNA and detect mainly the homologous mRNA. Thus, the noncod- and transfer analysis of human mRNA. The mouse exon 2 ing regions of the two MBP mRNAs have diverged signifi- probe detected bands of the appropriate size in mouse DNA, cantly, while the coding regions have been conserved. but it hybridized to a repeat sequence in human genomic Nucleotide Sequence of Human MBP cDNA. The 2.2-kb digests. However, the probe did recognize a human mRNA human MBP cDNA was sequenced by a combination of the of2.2 kb, the size ofauthentic human MBP message (data not dideoxy chain termination and chemical cleavage techniques. shown). The human brain stem/cerebellum cDNA library The cDNA sequence is shown in Fig. 2. There is a short, was then screened with this mouse exon 2 probe. Five 36-bp, 5' untranslated region followed by a coding sequence hundred thousand clones were screened and 17 positive of 516 bp and a long, 1603-bp, 3' untranslated region con- phages were identified. Six of these phages were plaque taining a polyadenylylation signal. The structure of the purified, and all hybridized to pHBP-1, the MBP coding human MBP mRNA is quite similar to that ofboth the rat and region probe, as well. mouse. The human coding region is greater than 90% homol- In the mouse the amino acid sequence encoded by exon 2 ogous to both rat and mouse coding regions. However, as was is present in both the 17.0- and 21.5-kDa proteins, the larger Downloaded by guest on September 26, 2021 4964 Neurobiology: Kamholz et al. Proc. Natl. Acad Sci. USA 83 (1986)

GAAAACAGTGCAGCCACCTCCGAGAGCCTGGATGTGATG GCG TCA CAG AAG AGA CCC TCC CAG AGG CAC GGA TCC AAG TAC CTG GCC ACA 90 MET Ala Ser Gln Lys Arg Pro Ser Gln Arg His Gly Ser Lys Tyr Leu Ala Thr 17

GCA AGT ACC ATG GAC CAT GCC AGG CAT GGC TTC CTC CCA AGG CAC AGA GAC ACG GGC ATC CTT GAC TCC ATC GGG CGC TTC 171 Ala Ser Thr MET Asp His Ala Arg His Gly Phe Leu Pro Arg His Arg Asp Thr Gly Ile Leu Asp Ser Ile Gly Arg Phe 44

TTT GGC GGT GAC AGG GGT GCG CCA AAG CGG GGC TCT GGC AAG GAC TCA CAC CAC CCG GCA AGA ACT GCT CAC TAT GGC TCC 252 Phe Gly Gly Asp Arg Gly Ala Pro Lys Arg Gly Ser Gly Lys Asp Ser His His Pro Ala Arg Thr Ala His Tyr Gly Ser 71 CTG CCC CAG AAG TCA CAC GGC CGG ACC CAA GAT GAA AAC CCC GTA GTC CAC TTC TTC AAG AAC ATT GTG ACG CCT CGC ACA 333 Leu Pro Gln Lys Ser His Gly Arg Thr Gln Asp Glu Asn Pro Val Val His Phe Phe Lys Asn Ile Val Thr Pro Arg Thr 98 CCA CCC CCG TCG CAG GGA AAG GGG AGA GGA CTG TCC CTG AGC AGA TTT AGC TGG GGG GCC GAA GGC CAG AGA CCA GGA TTT 414 Pro Pro Pro Ser Gln Gly Lys Gly Arg Gly Leu Ser Leu Ser Arg Phe Ser Trp Gly Ala Glu Gly Gln Arg Pro Gly Phe 125 GGC TAC GGA GGC AGA GCG TCC GAC TAT AAA TCG GCT CAC AAG GGA TTC AAG GGA GTC GAT GCC CAG GGC ACG CTT TCC AAA 495 Gly Tyr Gly Gly Arg Ala Ser Asp Tyr Lys Ser Ala His Lys Gly Phe Lys Gly Val Asp Ala Gln Gly Thr Leu Ser Lys 152 ATT TTT AAG CTG GGA GGA AGA GAT AGT CGC TCT GGA TCA CCC ATG GCT AGA CGC TGAAAACCCACCTGGTTCCGGAATCCTGTCCTCAG 584 Ile Phe Lys Leu Gly Gly Arg Asp Ser Arg Ser Gly Ser Pro MET Ala Arg Arg * 170 CTTCTTAATATAACTGCCTTAAAACTTTAATCCCACTTGCCCCTGTTACCTAATTAGAGCAGATGACCCCTCCCCTAATGCCTGCGGAGTTGTGCACGTAGTAGGGT 691 CAGGCCACGGCAGCCTACCGGCAATTTCCGGCCAACAGTTAAATGAGAACATGAAAACAGAAAACGGTTAAAACTGTCCCTTTCTGTGTGAAGATCACGTTCCTTCC 798 CCCGCAATGTGCCCCCAGACGCACGTGGGTCTTCAGGGGGCCAGGTGCACAGACGTCCCTCCACGTTCACCCCTCCACCCTTGGACTTTCTTTTCGCCGTGGCTCGG 905 CACCCTTGCGCTTTTGCTGGTCACTGCCATGGAGGCACACAGCTGCAGAGACAGAGAGGACGTGGGCGGCAGAGAGGACTGTTGACATCCAAGCTTCCTTTGTTTTT 1012 1119 1226 1333 1440 AnAAaGGTACGAGTCCTCTAGACAGCCTCGCAGCCGCGCCAGTCGCCCATAGACACTGGCTGTGACCGGGCGTGCTGGCAGCGGCAGTGCACAGTGGCCAGCACTAA 1547 1654 1761 ACCTTTTGATTTTAGCGCACCTGTGATTGATAGGCCTTCCAAAGAGTCCCACGCTGGCATCACCCTCCCCGAGGACGGAGATGAGGAGTAGTCAGCGTGATGCCAAA 1868 1975 2082 TGGCTGAACGGAAAGTGTACACTGTTCCTGAATATTGAAATAAAACAATAAACTTTTAAAAAAAAAAAAAA 2155 FIG. 2. Nucleotide sequence ofhuman MBP mRNA. The sequence was determined independently on both strands. The corresponding amino acid sequence of MBP is also shown.

of which also contains the amino acid sequence encoded by We have confirmed the existence of a human 21.5-kDa exon 6. We examined the 6 cDNA clones containing exon 2 MBP by probing blots of total human myelin with a polyclo- sequences to see if they also contained exon 6 sequences by nal antiserum raised against a synthetic polypeptide corre- Southern transfer hybridization using a synthetic oligonucle- sponding to part ofthe mouse exon 2-encoded sequence. This otide 25 bases long. All of these clones also contained exon serum identifies a minor human MBP of 21.5 kDa. Antisera 6 homologous sequences (data not shown). raised against a synthetic peptide corresponding to the first 20 One ofthe cDNA clones that contained exon 2 information amino acids ofMBP also recognize this 21.5-kDa protein (see and carried a 2.2-kb insert was subcloned and its coding Fig. 5). Antisera raised against a synthetic peptide encoded region was sequenced. The nucleotide sequence was identi- by mouse exon 6 did not recognize the 21.5-kDa human MBP. cal to that of the 18.5-kDa MBP cDNA, but it contained a The reason for this is not known; however, Kerlero de Rosbo 78-bp (26 amino acid) insertion beginning at nucleotide et al. (8), using a monoclonal that recognized the same epitope, also failed to detect this protein. The cDNAs position 214 (amino acid 58) relative to the 18.5-kDa MBP authentic a of 21.5 identified with the mouse exon 2 probe are, thus, cDNA. The cDNA, thus, encodes putative MBP mRNAs directing the synthesis of MBP proteins, and not kDa. The 78-bp (26 amino acid) N-terminal insertion is the merely unspliced precursors. No cDNA or protein homolo- same size and occurs at the same position within the protein gous to the mouse 17.0-kDa (pre-small) MBP has been as that found in the 17.0- and 21.5-kDa mouse MBPs. The detected in human myelin. nucleotide and amino acid sequences of mouse exon 2 are Identification ofa cDNA Encoding a 17.2-kDa MBP. Deibler compared to their human counterparts in Fig. 4. The se- et al. have recently identified a 17.2-kDa human MBP (23). quences are identical in 22 of26 amino acids. There have been This isoform is identical to the 18.5-kDa MBP but is missing 5 single base pair changes, two within a single codon, and two amino acids 106-116 ofthe larger protein. The corresponding of the four amino acid changes are not conservative. Both cDNA would, thus, be missing nucleotides 354-387 of the sequences contain a cysteine, a residue not found in the coding region sequence shown in Fig. 2 and could be 18.5-kDa MBP. The presence of this human cDNA that identified by the absence of a characteristic 210-bp Dde I contains extra sequences equivalent to a mouse exon sug- fragment arising from nucleotide positions 372-581. DNAs gests that the human gene may have an exon arrangement from 12 phages that had been shown to contain MBP similar to that of the mouse. sequences during the initial screening were cleaved with Downloaded by guest on September 26, 2021 Neurobiology: Kamholz et al. Proc. Natl. Acad. Sci. USA 83 (1986) 4965 E B H E B H A B C

233-9206EJ1 be 25.0- 44- ttW

23 204- .

18.0- U-

14.0-

FIG. 5. Immunoblot analysis of human myelin. (A) Rabbit anti- body to human MBP N terminus; (B) Rabbit antibody to mouse MBP exon 6; (C) Rabbit antibody to mouse MBP exon 2. Calibration on left is in kDa. FIG. 3. Southern blot analysis of human placental DNA. (Left) pHBP-1 probe; (Right) pHBP-2 probe. E, EcoRI; B, BamHI; H, HindIII. The minor bands seen in Right (lanes B and H) are due to 18.5-kDa protein is the major human MBP, and a protein of incomplete removal of the pHBP-1 probe from the filter prior to similar size and amino acid sequence is found in myelin of reprobing with pHBP-2 sequences. The upper band seen in lane E is several other vertebrate species (7, 8). A 21.5-kDa MBP a partial digestion product. Ten micrograms of DNA was loaded per isoform has been lane and the filter was exposed to x-ray film with an intensifying detected in mouse (21), rat (22), sheep (18), screen for 16 hr at -70'C. Positions of marker fragments (kb) are cow (19), rabbit (20), and guinea pig (our unpublished indicated on the left. results), and it has been detected in human myelin by some workers (7) but not others (8). The 17.2-kDa protein is similar EcoRI and Dde I, and the resultant fragments were separated in size to the mouse 17.0-kDa (pre-small) MBP but differs on agarose gels, transferred to nylon, and probed with from this protein in amino acid sequence. A putative 17.2- pHBP-1. Two phage DNAs were found that were missing the kDa MBP homologue has been detected in several other 210-bp Dde I band. One of these, which contained a 1.1-kb species (8), and a mouse cDNA that may encode a similar cDNA, was partially sequenced. The coding region of this protein has been identified (F.d.F. and J.K., unpublished cDNA was identical to the 18.5-kDa MBP cDNA but was results). missing nucleotides 354-387 encoding amino acids 106-116. The simple pattern offragments seen on genomic digests of This cDNA corresponds to the 17.2-kDa protein described by human DNA after hybridization with MBP cDNA probes Deibler et al. The missing amino acids exactly correspond to suggests there is a single MBP gene in the . mouse exon 5, which again suggests that the human and Use of the human MBP cDNA has recently allowed identi- mouse exon arrangements are quite similar forthe MBP gene. fication of restriction fragment length polymorphisms A comparison of the structures of the multiple mouse and (RFLPs), which segregate as simple Mendelian traits in the human MBP mRNAs is shown in Fig. 6. human population, supporting this assertion (R. Spielman, J.K., and R.L., unpublished data). The sequences added to DISCUSSION form the 21.5-kDa MBP and deleted to produce the 17.2-kDa The preceding data show that there are at least three separate MBP correspond exactly to mouse exons 2 and 5. Thus, the human MBP mRNAs, which encode proteins of 21.5, 18.5, human MBP gene shares two of the seven mouse exons and and 17.2 kDa. These three proteins share a common sequence is likely to have an exon arrangement similar to that ofmouse. and differ by the insertion of a 26-residue amino acid These data suggest that the three human MBPs are, as in the sequence in the 21.5-kDa isoform or by deletion of an mouse, formed from alternative splicing of a primary human 11-residue amino acid sequence in the 17.2-kDa isoform. The MBP transcript. Other mechanisms, such as the existence of 1 2 3 4 5 6 7 8 9 10 11 12 13 HUMAN Val Pro Trp Leu Lys Pro Gly Arg Ser Pro Leu Pro Ser GTA CCC TGG CTA AAG CCG GGC CGG AGC CCT CTG CCC TCT -__------A- A------MOUSE Val Pro Trp Leu Lys Glu Ser Arg Ser Pro Leu Pro Ser

14 15 16 17 18 19 20 21 22 23 24 25 26 HUMAN His Ala Arg Ser Gln Pro Gly Leu Cys Asn MET Tyr Lys CAT GCC CGC AGC CAG CCT GGG CTG TGC AAC ATG TAC AAG -GT C-- MOUSE His Ala Arg Ser Arg Pro Gly Leu Cys His MET Tyr Lys

FIG. 4. Comparison of mouse and human exon 2 sequences. Downloaded by guest on September 26, 2021 4966 Neurobiology: Kamholz et al. Proc. Natl. Acad Sci. USA 83 (1986)

Mouse MBP Isoforms Human MBP Isoforms kDa 1 2 3,4 5 6 7 kDa 1 2 3,4 5 6 7 21.5 1 Ad 21.5 E 1a3 I 3,4 5 6 7 1 3,4 5 6 7 18.5 18.5 [ I_ 1 2 3,4 5 7 1 3,4 6 7 17.0 1 aIN 1 17.2 1 1 _ 1 3,4 5 7 14.0 I FIG. 6. Comparison of mouse and human MBP isoforms. The numbers above the bars refer to the exons from the mouse MBP gene and their presumptive human counterparts. multiple MBP alleles or a duplication ofthe MBP gene, might Lynn Hudson for help with immunoblotting and Judy Hertler and also be considered, but they are much less likely. Charlene French for expert clerical assistance. We especially ac- Mouse myelin contains two MBPs, a 14.0- and a 17.0-kDa knowledge the cooperation ofDr. Lucy Rorke ofChildren's Hospital protein, which are not present in the human. Both of these ofPhiladelphia, without whose help we could not have obtained such mouse proteins are missing amino acids 117-158 of the excellent human brain material. 18.5-kDa MBP, which correspond to exon 6 of the mouse 1. Braun, P. E. (1984) in Myelin, ed. Morrell, P. (Plenum, New MBP gene. No human MBP missing these residues has been York), pp. 97-116. found to date. An attractive explanation for these differences 2. Sternberger, N. H., Itoyama, Y., Kies, M. & Webster, is that the equivalent human exon 6 sequences are not H. deF. (1978) Proc. Natl. Acad. Sci. USA 75, 2521-2524. alternatively spliced. Were this the case, the human MBP 3. Hartman, B. K., Agrawal, H. C., Agrawal, D. & Kalmbach, repertoire would not include proteins lacking these se- S. (1982) Proc. Natl. Acad. Sci. USA 79, 4217-4220. quences. Several explanations for this difference in splicing 4. de Ferra, F., Engh, H., Hudson, L., Kamholz, J., Puckett, C., pattern between humans and mice are possible. However, a Molineaux, S. & Lazzarini, R. A. (1985) 43, 721-727. and human MBP genes and their 5. Takahashi, N., Roach, A., Teplow, D. B., Prusiner, S. B. & comparison of the mouse Hood, L. (1985) Cell 42, 139-148. intron-exon boundary sequences may help clarify this issue. 6. Carnegie, P. R. (1971) Biochem. J. 123, 57-67. The human MBP mRNA, like that in the rat and mouse, 7. Waehneldt, T. V., Malotka, J., Karin, N. J. & Matthieu, J.-M. contains an ORF in the 3' untranslated portion. The mouse 3' (1985) Neurosci. Lett. 57, 97-102. ORF contains an in-frame consensus splice sequence, which 8. Kerlero de Rosbo, N., Carnegie, P. R., Bernard, C. C. & could be spliced into an upstream splice donor. Such alter- Linthicum, D. S. (1984) Neurochem. Res. 9, 1359-1369. native splicing would yield an mRNA encoding a MBP with 9. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, an altered C terminus. The second ORF present in human W. J. (1979) Biochemistry 18, 5294-5299. MBP transcripts is a less likely candidate for alternative 10. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) in Molec- that are ular Cloning: A Laboratory Manual (Cold Spring Harbor splicing, since it does not contain in-frame sequences Laboratory, Cold Spring Harbor, NY). good matches with the consensus sequence for splice sites. 11. Huynh, T. V., Young, R. A. & Davis, R. W. (1984) in DNA The functional significance of multiple MBPs in any spe- Cloning Techniques: A Practical Approach, ed. Glover, D. cies is not understood. However, the conservation of the (IRL, Oxford). mouse exon 2 sequence in the human 21.5-kDa isoform and 12. Puckett, C., Kamholz, J., Rorke, L. & Lazzarini, R. A. (1985) the existence of a 21.5-kDa MBP in several other species as Neurology 35, Suppl. 1, 259. well suggest that this 26-residue amino acid sequence has an 13. Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J. H. & function. Since it is known that MBP is located on Roe, B. A. (1980) J. Mol. Biol. 143, 161-178. important 14. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, the inner cytoplasmic surface of the mem- 499-560. brane, this sequence may interact with myelin or other 15. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. proteins to direct MBP to the correct position for the Acad. Sci. USA 76, 4350-4354. initiation of myelination. After initiation, the other isoforms 16. Richardson, C., Berkovich, A., Rozenblatt, S. & Bellini, W. J. of MBP may be capable of self-aggregation to complete (1985) J. Virol. 54, 186-193. amounts of the 21.5- or 17. Gibson, B. W., Gillion, R. D., Whitaker, J. N. & Biemann, K. myelin assembly. Thus, only minor (1984) J. Biol. Chem. 259, 5028-5031. 17.0-kDa MBP would be necessary. Although no data are 18. Carnegie, P. R. & Dowse, C. A. (1983) Science 223, 936-938. currently available to prove this hypothesis, it has several 19. Reidl, L. S., Campagnoni, C. W. & Campagnoni, A. T. (1981) readily testable consequences. Alternative splicing of the J. Neurochem. 37, 373-380. MBP RNA, leading to the production of multiple MBPs may, 20. Agrawal, H. C., Randle, C. C. & Agrawal, D. (1981) J. Biol. Chem. 256, 12243-12246. thus, be important for the assembly and maintenance of the 21. Barbarese, E., Braun, P. E. & Carson, J. H. (1977) Proc. Natl. complex myelin membrane. Acad. Sci. USA 74, 3360-3364. 22. Agrawal, H. C., O'Connell, K., Randle, C. L. & Agrawal, D. The authors thank Susan Molineaux and David Nelson for daily (1982) Biochem. J. 201, 39-47. advice and discussions, George Englund, Bill Bellini, and Keith 23. Deibler, G., Krutzsch, H. & Kies, M., J. Neurochem., in McKenney for advice on DNA sequencing, Ward Odenwald and press. Downloaded by guest on September 26, 2021