Proc. Natl. Acad. Sci. USAVol. 86, pp. 2463-2467, April 1989Neurobiology

Cloning of a cDNA encoding the rat high molecular weightneurofilament peptide (NF-H): Developmental and tissueexpression in the rat, and mapping of its humanhomologue to chromosomes 1 and 22

(neurofilaments/intermediate ifiaments/transcription rates/chromosome mapping)

IVAN LIEBERBURG*t, NANCY SPINNERS, SUSAN SNYDER*, JOHN ANDERSON*, DMITRI GOLDGABER§,MILDRED SMULOWITZ¶, ZEV CARROLL¶, BEVERLY EMANUEL0, JOHN BREITNER*, AND LEE RUBINII*Fishberg Center for Neurobiology and Department of Psychiatry, Mount Sinai School of Medicine, New York, NY 10029; tDepartment of Genetics,Children's Hospital of Philadelphia, Philadelphia, PA 19104; §Department of Psychiatry, State University of New York, Stonybrook, NY 11794;IDepartment of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461; and I'Department of Pathology, College of Physicians andSurgeons, Columbia University, New York, NY 10032

Communicated by D. Carleton Gajdusek, October 10, 1988 (received for review July 25, 1988)

ABSTRACT Neurofflaments (NFs) are the intermediatefilaments specific to nervous tissue. They are probably essentialto the tensile strength of the neuron, as well as to transport ofmolecules and organelles within the axon. Three peptides withapparent molecular masses of approximately 68 (NF-L), 145(NF-M), and 200 (NF-H) kDa appear to be the major compo-nents of NF. The expression of these peptides is specific tonervous tissue and is developmentally regulated. Recently,complete cDNAs encoding NF-L and NF-M, and partial cDNAsencoding NF-H, have been described. To better understand thenormal and pathophysiology ofNFs we chose to clone the cDNAencoding the rat NF-H peptide. Using monoclonal antibodiesthat recognized NF-H, we screened a rat brain Agtll libraryand identified a clone that contained a 2100-nucleotide cDNAinsert representing the carboxyl-terminal portion of the NF-Hprotein. Anti-fusion protein antibodies recognized the NF-Hpeptide on immunoblots and stained fibrillar structures only inneurons. The cDNA recognized a 4500-nucleotide polyadenyl-ylated mRNA that was present only in nervous tissue and a3500-nucleotide mRNA in adrenal. Brain NF-H mRNA levelswere tightly developmentally regulated and paralleled thelevels ofNF-H peptide on immunoblots. Nuclear runoff studiesshowed that the 20-fold developmental increase in the NF-Hmessage was due only in part to a 4-fold increase in itstranscription rate. Levels of NF-H mRNA varied 20-foldamong brain regions, with highest levels in pons/medulla,spinal cord, and cerebellum, and lowest levels in olfactory bulband hypothalamus. Transcription studies revealed only a 2-folddifference in the transcription rates among these brain regions.Based on these results, we infer that half of the developmentalincrease and most of the interregional variation in the levels ofthe NF-H mRNA are mediated through message stabilization.Sequence information revealed that the carboxyl-terminalregion ofthe NF-H peptide contained a unique serine-, proline-,alanine-, glutamic acid-, and lysine-rich repeat. The serineresidues are likely sites of phosphorylation in the maturepeptide. Genomic blots revealed a single copy of the gene in therat genome and two copies in the human genome. In situhybridizations performed on human chromosomes mapped theNF-H gene to chromosomes 1 and 22. Whether one copy is apseudogene remains to be determined.

they assume 10-nm-diameter structures within the neuro-plasm (1-6). NFs are composed of three major polypeptides,which can easily be resolved by a variety of separationprocedures. On NaDodSO4/PAGE, the mammalian NF pep-tides migrate with apparent molecular masses of 195-220 kDa(NF-H), 140-160 kDa (NF-M), and 68-73 kDa (NF-L) (1-6).The variations in molecular mass are in part species depen-dent, but they may also reflect subtle differences in tech-nique. The peptides are variably phosphorylated, mainly onserine residues, with up to 22, 9, and 3 mol of phosphate,respectively, per mol of protein in the rat. Their degree ofphosphorylation may be regulated by development, neuronlocation within the central nervous system, and intraneuronalmicroenvironment, and they may be essential for intraneu-ronal assembly of the NF network (see refs. 1-6 and refs.therein).

In many central nervous system disorders, such as Alz-heimer disease, amyotrophic lateral sclerosis, toxic neurop-athies, and dementia pugilistica, there is histologic evidencefor disruption of normal NF transport and structure (7). Tobetter understand the nature of these illnesses, it is essentialto obtain molecular probes for these proteins. Recently,cDNAs encoding the entire protein sequences ofNF-L (8, 9)and NF-M (10) have been described. During the course ofthiswork, two independent groups cloned the murine gene (9) anda partial rat cDNA (11) encoding NF-H by cosmid walkingand immunologic screening, respectively.

In this paper, we describe a cDNA that encodes thecarboxyl-terminal tail of the rat NF-H peptide and use it tostudy developmental regulation, tissue-specific expression,and brain region expression ofthe NF-H gene.** In addition,we have mapped its human homologue to chromosomes 1 and22. Some of these results have been published in abstractform (12).

MATERIALS AND METHODScDNA Cloning. Cloning of the cDNA encoding NF-H was

accomplished by using mouse monoclonal antibodies (mAbs)to screen a Agtll library constructed from adult rat brainmRNA (kindly provided by David Anderson, Division of

Neurofilaments (NFs) are complex oligomeric peptide struc-tures that form the internal matrix of neurons. They areclassified into the category of intermediate filaments, in that

Abbreviations: NF, neurofilament; NF-H, 200-kDa NF peptide;NF-M, 145-kDa NF peptide; NF-L, 68-kDa NF peptide; mAb,monoclonal antibody; IPTG, isopropyl ,3-D-thiogalactoside.tTo whom reprint requests should be sent at the present address:Athena Neurosciences, 800-F Gateway Boulevard, South SanFrancisco, CA 94080.**The sequence reported in this paper is being deposited in theEMBL/GenBank data base (accession no. J04517).

2463

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

2464 Neurobiology: Lieberburg et al.

Biology, California Institute of Technology). Anti-NF mAbsused have been described (13). mAbs designated NP-12 (ref.13; kindly provided by Peter Davies, Albert Einstein Collegeof Medicine) and SMI-32 (Sternberger-Meyer) recognizealkaline phosphatase-resistant epitopes on NF-M and NF-H(13). Filters were screened with mAb supernatants diluted 1:5or ascites antibodies diluted 1:100 or 1:500 and visualizedwith horseradish peroxidase-coupled second antibody. Pos-itives were lysogenized into Y1089 (14). Fusion protein wasinduced in liquid cultured lysogens with isopropyl f-D-thiogalactoside (IPTG) induction (14). Anti-fusion proteinantibodies were raised in individual rats by injecting boiledand lysed bacterial pellets derived from 5-ml lysogen cultures(14). Alum was mixed 1:1 with the bacterial lysate as anadjuvant. Rats were given booster injections three additionaltimes at 3-week intervals and then bled. Whole rat sera wereused on immunoblots (see below) at a 1:1000 dilution.NaDodSO4/Gel Electrophoresis and Immunoblotting. NFs

were purified from rat brain (Sprague-Dawley, Charles RiverBreeding Laboratories) as described (1). Total rat brainprotein was prepared by direct homogenization of tissue intoNaDodSO4 sample buffer (15). Proteins were separated on7.5% NaDodSO4/polyacrylamide gels and blotted electri-cally (15). Probing, washing, and visualizing of immunoblotswere performed as described (15).

Preparation and Analysis of RNA and DNA. RNA wasprepared from total brain or dissected regions (16) by theguanidinium thiocyanate/LiCl procedure and separated onformaldehyde gels prior to blotting (17). Rat liver and humanplacenta genomic DNA were prepared, cut, and blottedaccording to Southern (17). DNA sequencing was accom-plished by the dideoxynucleotide termination method (17).Probes were labeled by nick-translation to a final specificactivity of 5 x 108-109 cpm/,Ag (17). All Northern andSouthern hybridizations were performed at 420C in a solutioncontaining 50% formamide, 5 x SSC (20x SSC = 3 MNaCl/0.3 M sodium citrate, pH 7.0), 5 x Denhardt's solution(17), 0.1% NaDodSO4, 50 mM sodium phosphate (pH 6.5), 2mM EDTA, sheared salmon sperm DNA (250 ,ug/ml), andyeast tRNA (250 ,ug/ml), using a probe concentration of 10ng/ml. Blots were washed to a final stringency of 0.1xSSC/0.1% NaDodSO4 at 55°C. In situ hybridizations to humanchromosomes were accomplished as described (18). Meta-phase spreads were selected by criteria of good chromosomemorphology and limited background grains. Chromosomallocation of grains was analyzed by microscopic observation.

Nuclear Runoff Assays. Nuclear runoff experiments wereconducted on whole rat brain cell nuclei obtained fromembryonic day 20 and adult day 70 animals or from adult ratbrain regions dissected according to McEwen et al. (16).Brain cell nuclei were freshly prepared on sucrose stepgradients (17), labeled with [a-32P]UTP (3000 Ci/mmol; 1 Ci= 37 GBq), and the RNA was prepared according to Green-berg and Ziff (19). Measurement of labeled runoff RNA wasperformed on triplicate filters according to Spindler et al.(20). Filters contained 5 jig of single-stranded M13 with theNF-H cDNA insert. A filter containing the sense strand ofthecDNA served as an internal control for the antisense M13filter. Hybridization efficiency (average 25 ± 5%) was esti-mated by the addition of trace amounts of sense 3H-labeledcomplementary RNA that had been generated from SP6transcriptions of the NF-H cDNA. Alternatively, labeledrunoff transcripts were visualized on slot or dot blots (19).

RESULTS

Initially, 100,000 plaques were screened, and 2 positives wereobtained. One clone produced a fusion protein that reacted

strongly with mAbs NP-12 and SMI-32, but not with controlmAbs or any anti-NF-H mAbs that recognized phosphatase-

sensitive epitopes (13). The clone was lysogenized into Y1089,and fusion protein was induced with 5 mM IPG. On immu-noblots of lysogen lysates probed with SMI-32 or NP-12, aprimary fusion protein band was identified with a molecularmass of 4185 kDa, as well as several smaller bands. No fusionprotein was induced in the absence of IPIG.To confirm the identity of this cDNA clone, we prepared

anti-fusion protein antibodies as described by Ravetch et al.(14). These antibodies were used to probe immunoblots of astandard NF preparation or of total rat brain protein. Pre-immune sera and non-IPTG-induced lysate antisera did notlabel any bands (data not shown). However, IPTG-inducedlysate antiserum clearly recognized NF-H, but not NF-M orNF-L. On immunoblots of total rat brain protein, we saw aprominent 200-kDa peptide but also could occasionally iden-tify a 160-kDa band, which probably represents the unphos-phorylated form of NF-H (3, 4). The anti-fusion proteinantiserum was also able to specifically stain fibrillar struc-tures in the soma and processes ofneurons in a pattern similarto that seen with NP-12 or SMI-32 (data not shown).The A clone contained a 2100-base-pair cDNA insert,

which, upon sequencing, contained a poly(A) tail and apolyadenylylation signal (AATAAAA) at one end of theinsert. The sequence from the other end contained a singleopen reading frame that translated to the amino acid se-quence depicted in Fig. 1, revealing a prevalent six-aminoacid repeat. This sequence was also seen in the two previ-ously mentioned NF-H cloning studies (9, 11).Using the cDNA as a probe, we identified a single,

4500-nucleotide, polyadenylylated RNA that was presentonly in nervous tissue and a 3500-nucleotide RNA presentonly in adrenal (Fig. 2). The levels ofNF-H mRNA increaseddramatically (20-fold) with age and paralleled the levels of theNF-H peptide on immunoblots of total brain protein (Fig. 2).To determine whether this increase was due to transcriptionalactivation, we carried out nuclear runoff assays on rat brainnuclei. These studies revealed a 4-fold increase in thetranscription rate of the NF-H gene between embryonic day20 (5 ± 2 ppm) and adult day 70 (19 ± 3 ppm). NF-H mRNAlevels varied 20-fold among adult rat brain regions, withhighest levels in pons/medulla, spinal cord, and cerebellum,and lowest levels in olfactory bulb, hypothalamus, andparietal cortex (Fig. 2). Nuclear runoff studies revealed onlya 2-fold difference in NF-H transcription rates by dot/slotblot analysis (19) among these brain regions, with highertranscription rates in the brain regions that contained abun-dant NF-H mRNA (i.e., pons/medulla, cerebellum, andspinal cord; data not shown).Genomic Southern analysis revealed that the rat genome

contained a single copy of the NF-H gene. Since the mAbsused in our studies have been shown to react with NF-H inmany mammalian species, including humans (14), we usedthe NF-H cDNA to probe human RNA and genomic South-ern blots under high stringency. Under such conditions, wealways observed a single 4500-nucleotide message on North-ern blots containing RNA obtained from human cerebralcortex or cerebellum (data not shown). Analysis of humanSouthern blots under identical conditions demonstrated thatthe human genome contains at least two copies of the NF-Hgene, as all restriction enzymes tested produced at least twomajor bands (Fig. 3). We have extended this analysis of thehuman genome to 35 infrequently cutting restriction enzymeswith similar results (I.L., P. St. George-Hyslop, and J.Gusella, unpublished results). The presence of at least twocopies of the NF-H gene has been confirmed by in situhybridization. Three independent in situ hybridization ex-periments were performed with normal human metaphasechromosomes and the rat NF-H probe. A total of 131metaphase spreads were analyzed in which 288 grains wereon chromosomes. In all experiments, there were two peaks

Proc. Natl. Acad. Sci. USA 86 (1989)

Neurobiology: Lieberburg et al.

54CCA GCT GAA GTC AAA TCT CCA GCT GAG GTC AAA TCT CCA GCT GAG GCC AAG TCAPro Ala Glu Val Lys Ser Pro Ala Glu Val Lys Ser Pro Ala Glu Ala Lys Spe

108CCA GCT GAG GCC AAG TCA CCA GCT GAG GTC AAA TCT CCA GCT ACA GTG AAG TCTPro Ala Glu Ala Lys Ser. Pro Ala Glu Val Lys Ser Pro Ala Thr Val Lys Ser

162CCA GGT GAG GCC AAG TCC CCA GCT GAG GCC AAG TCA CCA GCT GAG GTC AAA TCTPro Gly Glu Ala Lys Ser pro Ala Glu Ala Lys Ser Pro Ala Glu Val Lyc Rer

216CCA GTG GAG GCC AAG TCA CCA GCT GAG GCC AAG TCT CCA GCT TCA GTG AAG TCCPro Val Glu Ala Lys SerP Ala Glu Ala Lys Ser Pro Ser Val Lys Ser

270CCA GGT GAG GCC AAG TCA CCA GCT GAG GCC AAG TCA CCA GCT GAG GTC AAA TCTpro Gly Glu Ala Lys Spr Pro Ala Glu Ala Lys Ser Pro Ala Glu Val LysS-eir

324CCA GCT ACA GTG AAG TCC CCA GTT GAG GCC AAG TCA CCA GCT GAG GTC AAA TCTPro Al.a Thr Val Lys Ser Pro Val Glu Ala Lys Ser Pro Ala Glu Val LysZSer

378CCA GTT ACA GTG AAG TCC CCA GCT GAG GCC AAG TCA CCA GTT GAG GTC AAA TCTPro Val Thr Val Lys SAer Pro Ala Glu Ala Lys Ser Pro Val Glu Val Lys Rer

432CCA GCT TCG GTG AAG TCC CCA AGT GAA GCC AAG TCA CCA GCT GGA GCC AAG TCApro Ala Ser Val Lys Ser Pro Ser Glu Ala Lys Ser Pro Ala Gly Ala LYs Ser

486CCA GCT GAG GCC AAG TCA CCA GTT GTG GCC AAA TCA CCA GCT GAG GCC AAG TCAPro Ala Glu Ala Lys Ser Pro Val Val Ala Lys Ser Pro Ala Glu Ala LysSer

540CCA GCT GAG GCC AAG CCT CCA GCT GAG GCC AAG TCA CCA GCT GAG GCC AAG TCTPro Ala Glu Ala Lys Prn Prn Ala Glu Ala Ly Sr ProAgla Glu Ala Lys Ser

594CCA GCT GAG GCC AAG TCT CCA GCT GAG GCC AAG TCA CCA GCT GAG GCC AAG TCAPro Ala Glu Ala Lys Ser Po Ala Glu Ala Lys Ser Pro Ala Glu Ala Ly Sepr

648CCT GTT GAG GTA AAA TCT CCA GAG AAG GCC AAG AGC CCC GTG AAG GAA GGT GCAPro Val Glu Val Lys Ser Pro Glu Lys Ala Lys Ser Pro Val Lys Glu Gly Ala

702AAA TCC CTA GCT GAG GCC AAG TCC CCT GAG AAG GCC AAG TCC CCT GTG AAG GAALys Ser Leu Ala Glu Ala Lys Ser Pro Glu Lys Ala Lys SSer Pro Val Lys Glu

756GAG ATC AAG CCT CCA GCT GAG GTG AAA TCC CCC GAG AAG GCC AAG AGC CCC ATGGlu Ile Lys Pro Pro Ala Glu Val Lys Ser Pro Glu Lys Ala Lys Ser Pro MET

810AAG GAG GAG GCC AAG TCT =OO CAC AAC GCC AAG ACT CTG GAT GTn AAG TCT CGALys Glu Glu Ala Lys Ser Pro Glu Lys Ala Lys Thr Leu Asp Val Lys Ser Pro

864GAA GCC AAG ACT CCA GCG AAG GAG GAA GCA AAG CGC CCC GCA GAC ATC AGA TCCGlu Ala Lys Thr Pro Ala Lys Glu Glu Ala Lys Arg Pro Ala Asp Ile Arg Ser

918CCT GAG CAG GTC AAA AGT CCT GCC AAG GAG GAG GCC AAG TCC CCC GAG AAG GAAPro Glu Gln Val Lys Ser Pro Ala Lys Glu Glu Ala Lys Ser Pro Glu Lys Glu

972GAG ACC AGG ACT GAA AAG GTG GCT CCC AAG AAG GAA GAG GTG AAG TCC CCT GTGGlu Thr Arg Thr Glu Lys Val Ala Pro Lys Lys Glu Glu Val Lys Ser Pro Val

1026GAG GAG GTA AAA GCC AAA GAA CCC CCA AAG AAG GTG GAG GAG GAG AAG ACA CCAGlu Glu Val Lys Ala Lys Glu Pro Pro Lys Lys Val Glu Glu Glu Lys Thr Pro

1080GCC ACA CCA AAG ACA GAG GTG AAG GAG AGC AAG AAA GAT GAA GCT CCC AAG GAGAla Thr Pro Lys Thr Glu Val Lys Glu Ser Lys Lys Asp Glu Ala Pro Lys Glu

1134GCC CAG AAG CCC AAG GCG GAG GAG AAG GAG CCT CTC ACA GAA AAG CCC AAG GACAla Gln Lys Pro Lys Ala Glu Glu Lys Glu Pro Leu Thr Glu Lys Pro Lys Asp

1188TCT CCG GGG GAA GCC AAG AAG GAA GAG GCT AAA GAG AAG AAG GCG GCG GCC CCASer Pro Gly Glu Ala Lys Lys Glu Glu Ala Lys Glu Lys Lys Ala Ala Ala Pro

1242GAG GAG GAG ACG CCC GCC AAG TTG GGC GTG AAA GAA GAG GTC AAA CCC AAA GAGGlu Glu Glu Thr Pro Ala Lys Leu Gly Val Lys Glu Glu Val Lys Pro Lys Glu

1296AAG GCA GAA GAC GCC AAG GCC AAA GAA CCT AGC AAA CCC TCA GAG AAG GAG AAALys Ala Glu Asp Ala Lys Ala Lys Glu Pro Ser Lys Pro Ser Glu Lys Glu Lys

1350CCG AAG AAG GAG GAG GTG CCG GGC AGC ACA GAG AAG AAA GAC ACC AAG GAG GAGPro Lys Lys Glu Glu Val Pro Gly Ser Thr Glu Lys Lys Asp Thr Lys Glu Glu

1404AAG ACT ACG GAG TCC AAG AAG CCT GAG GAG AAA CCC AAA ATG GAG GCC AAG GCCLys Thr Thr Glu Ser Lys Lys Pro Glu Glu Lys Pro Lys MET Glu Ala Lys Ala

1458AAG GAG GAG GAC AAG GGC CTT CCC CAA GAG CCT AGC AAA CCC AAG ACA GAA AAGLys Glu Glu Asp Lys Gly Leu Pro Gln Glu Pro Ser Lys Pro Lys Thr Glu Lys

1512GCT GAA AAG TCC TCT AGC ACA GAC CAA AAA GAC AGC CAG CCC TCA GAG AAG GCCAla Glu Lys Ser Ser Ser Thr Asp Gln Lys Asp Ser Gln Pro Ser Glu Lys Ala

1566CCA GAG GAC AAG GCT GCC AAG GGA GAC AAG TAA GAG GAC GAG AGG GAC ACC CAGPro Glu Asp Lys Ala Ala Lys Gly Asp Lys

AAT AGC CAA AGA AAC TCA GGA CGG CCC CGG TAC TCA AGG GTT GGO GTA ATA AAGTTT ATT TCT TCC TTT CCC TCC GTA AGA AGA AAC ACC AAC TTA GAT GGC GGG CCCICC TCA OCA AAC AGO AAT TTC TAT TAG OAT TAA OTT AGO AAG AGA AlA TAA CCCTIA 0CC CTG OCT CCC CAA CAC CIA AAG CCC TOO CCA GOT OAT GIA CAA TTA TGATAGCTT ATC GTAGCC GAA CGTGATGTA TTG CTG AAC GCTCCA CGT AAA ACG CGTGAC TAA AAACTG COO CCCCTC CTT TCC AAG TAAGTG CAT TCACTT CCCGTA TGTCCT ACC GAC AGG TGA CCG CAG TAA TGA ATG AGC AGT TAG AAA TGC ATT ATG CTTGAA ATG TTG TAA CCT ATA CCCGAA TGC CTTCTTGTT TTC CAA AGG AGC GGT CAGGCCCTTGCCCGG TAC ACG CTC CTG GAA GAGCTG CAG CAG GTG AGG CAGGGG TGGCCG CTG AAC CAG GCC AGGGTG TGC TGTCCACTG AAG TCC ACT TTCGAT TGC TTCCGT GCA ATA AAACCA AGT GCT TCTGAA AAA AAA AAA AAA AAA

Proc. Natl. Acad. Sci. USA 86 (1989) 2465

0 U. 9F0 c h-.-I. O 2 X m-j

i. 0U)0 :E x

CO00w w 2 5 8

-28S

* *.911 14 17 20 30 50 70

Iw W -200 kd

a _ *S

-4.5 kb.

C b-

t,~~~~~~~~~U0 U

-4.5kb.

FIG. 2. The NF-H cDNA is nervous tissue specific, develop-mentally regulated, and varies greatly among brain regions. (Top) ANorthern blot containing total RNA (30 gg per lane) obtained fromthe indicated adult rat tissues. The blot was intentionally overex-posed for 2 weeks with an intensifying screen. In addition to the4500-nucleotide message in brain, note the presence of a 3500-nucleotide RNA in adrenal and the absence of detectable message inother tissues. (Middle) Total rat brain protein (100 Aug per lane) andtotal rat brain RNA (20 ,ug per lane) obtained from rats of increasingage from embryonic day 18 (lane E18) to adult day 70 (lane 70) wereprobed on immunoblots (Upper Middle) and Northern blots (LowerMiddle). Upper was probed with mAb NP-12 and Lower was probedwith nick-translated NF-H cDNA. Note the steady increase inimmunoreactive NF-H peptide, which is paralleled by the increasinglevels of the NF-H message. kd, Kilodaltons; kb, kilobases. (Bottom)A Northern blot containing total RNA (10 Ag per lane) obtained fromthe indicated adult rat brain regions probed with the NF-H cDNA.

of hybridization, one on chromosome 1 (1p21-lqll), with23%, 15%, and 14.3% of the total grains, and the second siteon the long arm of chromosome 22, with 18.5%, 15%, and18% of total grains. Chromosomal distribution of grains forthe three experiments is shown in Fig. 3.

DISCUSSIONIn this paper, we have described the cloning of a partialcDNA encoding the carboxyl-terminal tail of the rat NF-H

FIG. 1. Sequence of the NF-H cDNA clone. DNA sequence fromthe 5' end of the cDNA revealed a single open reading frame with theindicated amino acid sequence. A predominant amino acid repeatindicating probable phosphorylation sites (21) (Lys-Ser-Pro-Ala,Lys-Ser-Pro-Gly, or Lys-Ser-Pro-Val) is underlined. The oppositeend of the cDNA contained a poly(A) tail and a polyadenylylationsequence (AATAAAA).

2466 Neurobiology: Lieberburg et al.

v ao Es

0

23.1- 2

94-

4*-

20--

201510

5

za

L.0

aomz

20

15

10

5

p I q p2q q q lP q-~~~~ 4

6 7 8 9 0 Ii1 12

Pq p q Pq pqpqpqp'q q pqp q pq13 14 15 16 17 18 19 202122 X A

CHROMOSOMES

FIG. 3. The NF-H gene is represented by a single copy in the ratgenome and two copies in the human genome, which map tochromosomes 1 and 22. (Upper) Rat liver (Left) and human placenta(Right) (20 ,.g per lane) genomic DNA were cut with the indicatedrestriction enzymes, separated on 0.8% agarose gels, blotted, andprobed with the NF-H cDNA. Blots were washed to a finalstringency of0.1x SSC/0.1% NaDodSO4 at 550C prior to exposure.

The positions of A HindIll molecular mass markers are indicated (inkDa). Note the presence of single bands on the rat blot (Left)following digestion with each of the three enzymes, while on thehuman blot (Right) at least two bands are observed with every

enzyme. (Lower) In situ hybridization of the NF-H cDNA to normalhuman metaphase chromosomes. The histogram shows the distri-bution in 131 metaphase spreads hybridized with 3H-labeled NF-Hprobe. Abscissa represents chromosomes as they are observed on

G-banding; ordinate shows the number of silver grains.

peptide. A critical part of the screening process was the use

of mAbs that recognized phosphatase-insensitive epitopespresent on NF-H. Although there is limited protein sequence

information available for NF-H, we have a number of reasonsto believe that we have in fact cloned a cDNA encodingNF-H. (i) The anti-NF mAbs used for screening recognizedboth NF-M and NF-H on immunoblots; however, the anti-fusion protein antiserum recognized only NF-H but not

I.

NF-M on immunoblots of total rat brain protein or standardNF preparations. In addition, the anti-fusion protein anti-body specifically stained filaments present in soma andprocesses of cultured neurons in a pattern strikingly similarto that seen with anti-NF-H mAbs. The ability of an anti-fusion protein antiserum to recognize the original protein hadbeen used by Ravetch et al. (14) in confirming the identity ofa malarial cDNA clone. (ii) Our cDNA recognized a neuron-specific and developmentally regulated mRNA. The devel-opmental profile of the mRNA and the NF-H peptide corre-lated very well and agreed with previous reports of NF-Hpeptide expression. We were clearly able to detect the NF-HmRNA prior to birth. This is in agreement with the immuno-cytochemical results of Cochard and Paulin (22), althoughShaw and Weber (1) had not seen it at that stage. It is likelythat this discrepancy concerning the onset of NF-H expres-sion revolves around the different antibodies used by thevarious workers. (iii) The amino acid sequence predictedfrom our cDNA clone demonstrated a prevalence of certainamino acids that are known to occur in the NF peptides.Geisler et al. (23) have performed amino acid analysis of thecarboxyl-terminal region of the porcine NF-H peptide andfound that 80% of the residues were glutamic acid/glutamine,proline, glycine, alanine, serine, and lysine, and that thepreponderance of these amino acids was peculiar to thecarboxyl-terminal tails of the NF peptides. The single openreading frame of our cDNA predicts an amino acid sequencethat contains the appropriate ratio of those residues describedby Geisler et al. (23). (iv) Lastly, during the course of thiswork, using cosmid walking and immunologic screening,respectively, two independent groups cloned a murine ge-nomic fragment (9) and partial rat cDNAs (11) encodingNF-H. In both studies, probes from these clones identified asingle 4500-nucleotide message in the rodent brain, and in onereport a developmental profile similar to that reported here isdescribed (11). Also in that report, similar partial sequencedata as described here were reported (11).We found that the mRNA encoding NF-H was -4500

nucleotides long and would, therefore, be expected to encodemaximally a peptide of 165 kDa. Although NF-H has anapparent mass of 200 kDa on NaDodSO4 gels, its true massis closer to 112-138 kDa based on guanidine hydrochloridechromatography and sucrose density gradient centrifugation(2). Thus, the size of the mRNA is not inconsistent with thesize of the peptide. The smaller size of the adrenal NF-Htranscript (3500 nucleotides) deserves further investigationand may represent a splicing variant of the NF-H gene, sincePC-12 cells, a rat malignant pheochromocytoma line relatedto adrenal medullary cells, possess a 4500-nucleotide form ofthe NF-H message similar to that seen in brain (24).The repetitive sequence of amino acids within the car-

boxyl-terminal region of NF-H is unique; however, similarshort stretches can be found in the carboxyl-terminal tails ofNF-L (8) and NF-M (10). NF-H is phosphorylated exclu-sively on serine residues present in the carboxyl-terminal tailof the peptide (4). Based on an elegant immunologic study byLee and colleagues using synthetic peptide antisera (21), itseems very likely that the serines predicted by this sequenceare those that are phosphorylated in the mature peptide.The expression of the NF-H gene is nervous tissue specific

and developmentally regulated and presumably containsenhancer elements that determine these properties. Curi-ously, the 20-fold developmental increase in NF-H messageand peptide is mirrored by only a 4-fold increase in the gene'stranscription rate. By inference, we conclude that at least50% of the developmental increase in NF-H message levelsis achieved through message stabilization. Message stabili-zation has been shown to be important in a number ofsystems, including the regulation of NF-M in differentiatingPC-12 cells (24), and the autoregulation of 8-tubulin (25).

Proc. Natl. Acad. Sci. USA 86 (1989)

2C

A I AA I. . I I . a . . . I I I . . I I

Fc xla EA 8x LU is

Proc. Natl. Acad. Sci. USA 86 (1989) 2467

Interestingly, in PC-12 cells, although the levels of NF-Hprotein increase dramatically with nerve growth factor ad-ministration, the levels of NF-H message and transcriptionrates do not change (24). Thus, it would appear that there arenumerous regulatory points that can be used by the cell indetermining the amount of NF-H protein produced.Hoffman et al. (26) have recently proposed that NF gene

expression, at least in part, determines axon caliber. Theirstudy positively associated the expression ofNF-L with axoncaliber. Since the NF-H protein seems to be involved informing the noncovalent crosslinks between NFs in the axon(5), it is not surprising that NF-H levels are greatest inneurons that produce long thick axons. The Northern hy-bridization results presented here confirm this observation,indicating that brain regions giving rise to long-axon neurons(spinal cord, cerebellum, pons, medulla) contain high levelsof the NF-H message, as well as NF-H protein (data notshown). An unexpected result was that this interregionalvariation in NF-H mRNA levels is achieved largely throughmessage stabilization. f8-Tubulin message levels are regu-lated by the amount of unpolymerized tubulin monomerpresent in the cell, a property that is determined by the 5'untranslated portion of the ,8-tubulin message (25). Perhaps asimilar mechanism may be at play in adjusting NF-H messagelevels.

In many dementing illnesses, there appears to be a disrup-tion of normal NF transport and/or structure (7). In Alzhei-mer disease, the salient histologic feature is the presence ofneurofibrillary tangles and plaques associated with vacuolardegeneration and gliosis. The neurofibrillary tangles havebeen shown to contain bizarre structures termed pairedhelical filaments, which are NaDodSO4 and urea insoluble(7). There has been much debate concerning the constituentsof paired helical filaments and neurofibrillary tangles. Al-though these structures may contain unique epitopes, anti-bodies that recognize various neuronal proteins, includingthe NF peptides microtubule-associated protein (MAP)-2 andtau, crossreact with these structures. Recent evidence, usinga variety of techniques, suggests that tau, and not NFs orMAP-2, is a major component of these structures (see refs. 13and 27 and refs. therein). In addition, it has been demon-strated that many of the anti-NF mAbs that do recognizethese structures recognize phosphatase-sensitive epitopes onNFs and tau, and that anti-NF mAbs that are phosphataseinsensitive (NP-12 and SMI-32) do not react with tau ortangles (13). This suggests that the abnormal tau peculiar totangles may contain a general class of phosphorylatedepitopes that is normally present on a variety of neurofibril-lary proteins. One might infer from this the existence of asmall class of central nervous system kinases and phos-phatases that are constantly at play in modeling and remod-eling these complex neurofibrillary structures. Possibly, theetiology of Alzheimer disease is a dysfunction, either ac-quired or genetic, of one or more of these modifying en-zymes. Now that many of these filamentous proteins havebeen cloned, there exists the obvious potential for synthesisor expression of large quantities of the peptides for use assubstrates in isolating and characterizing these as yet uni-dentified modifying enzymes.While there is a single gene encoding the NF-H peptide in

the rat, there appear to be at least two NF-H genes in humans.At the present time, it is not known whether both of thesegenes are expressed, or if one of them is a pseudogene. Bothchromosomal locations to which the NF-H gene has beenmapped, the short arm of chromosome 1 and the long arm ofchromosome 22, are regions of the genome that have beenfound to be altered in tumors of neural origin. Deletions of lphave been observed frequently in neuroblastoma (28). There-are also other sequences in this region that have been shown

to be important in nerve cell growth and differentiation. TheN-ras oncogene has been mapped to 1p22 and/or 1p11-12,and the 8-subunit of nerve growth factor has been mapped to1p22.1 (29, 30). Abnormalities of chromosome 22 have beenobserved in a number of tumors of neuroectodermal origin.Loss or deletions of chromosome 22 (22qll-qter) have beenassociated with meningiomas and gliomas (31, 32), and arecent study demonstrated that sequences on chromosome 22are nonrandomly lost in acoustic neuromas (33). Reciprocaltranslocations involving chromosomes 11 and 22 have beenobserved in neuroepitheliomas (34) and in a neuroendocrinetumor of the small intestine (35). The involvement of theNF-H gene(s) in any of these pathological states remains tobe determined.

This work was supported by grants from the National Institutes ofHealth (I.L. and L.R.), The McKnight Foundation (I.L.), TheHartford Foundation (I.L.), The March of Dimes (L.R.), and TheMuscular Dystrophy Association (L.R.).

1. Shaw, G. & Weber, K. (1982) Nature (London) 298, 277-279.2. Kaufmann, E., Geisler, N. & Weber, K. (1984) FEBS Lett. 170, 81-84.3. Carden, M. J., Schlaepfer, W. W. & Lee, V. M. Y. (1985)J. Biol. Chem.

260, 9805-9817.4. Julien, J. P. & Mushynski, W. E. (1982) J. Biol. Chem. 257, 10467-10470.5. Hirokawa, N., Glicksman, M. & Willard, M. (1984) J. Cell Biol. 98,1523-

1536.6. Dahl, D. & Bignami, A. (1986) Exp. Cell Res. 162, 220-230.7. Gajdusek, D. C. (1985) New Engl. J. Med. 312, 714-719.8. Lewis, S. A. & Cowan, N. J. (1985) J. Cell Biol. 100, 843-850.9. Julien, J.-P., Meyer, D., Flavell, D., Hurst, J. & Grosveld, F. (1986) Mol.

Brain Res. 1, 243-250.10. Napolitano, E. W., Chin, S. S. M., Colman, D. R. & Liem, R. K. M.

(1987) J. Neurosci. 7, 2590-2599.11. Robinson, P. A., Wion, D. & Anderton, B. H. (1986) FEBS Lett. 209,

203-205.12. Lieberburg, I., Lappin, R., Davies, P. & Rubin, L. (1985) J. Cell Biol.

101, 17a.13. Ksiezak-Reding, H., Dickson, D. W., Davies, P. & Yen, S. H. (1987)

Proc. Nat!. Acad. Sci. USA 84, 3410-3414.14. Ravetch, J. V., Kochan, J. & Perkins, M. (1985) Science 227, 1593-1597.15. Lappin, R., Lieberburg, I. & Rubin, L. (1987) J. Neurobiol. 18, 75-99.16. McEwen, B. S., Plapinger, L., Wallach, G. & Magnus, C. (1972) J.

Neurochem. 19, 1159-1170.17. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1985) Molecular Cloning:A

Laboratory Manual (Cold Spring Harbor Laboratory, Cold SpringHarbor, NY).

18. Emanuel, B. S., Canizzaro, L. A., Seyer, J. M. & Meyers, J. C. (1985)Proc. Natl. Acad. Sci. USA 82, 3385-3389.

19. Greenberg, M. E. & Ziff, E. B. (1984) Nature (London) 311, 433-438.20. Spindler, S. R., Mellon, S. H. & Baxter, J. D. (1982) J. Biol. Chem. 257,

11627-11632.21. Lee, V. M.-Y., Otvos, L., Carden, M., Hollosi, M., Dietzschold, B. &

Lazzarini, R. A. (1988) Proc. Natl. Acad. Sci. USA 85, 1998-2002.22. Cochard, P. & Paulin, D. (1984) J. Neurosci. 4, 2080-2094.23. Giesler, N., Fischer, S., Vandekerckhove, J., VanDamme, J., Pless-

mann, U. & Weber, K. (1985) EMBO J. 4, 57-63.24. Lindenbaum, M. H., Carbonetto, S., Grosveld, F., Flavell, D. &

Mushynski, W. E. (1988) J. Biol. Chem. 263, 5662-5667.25. Gay, D. A., Yen, T. J., Lau, J. T. Y. & Cleveland, D. W. (1987) Cell 50,

671-679.26. Hoffman, P. N., Cleveland, D. W., Griffin, J. W., Landes, P. W.,

Cowan, N. J. & Price, D. L. (1987) Proc. Natl. Acad. Sci. USA 84, 3472-3476.

27. Goedert, M., Wischik, C. M., Crowther, R. A., Walker, J. E. & KMug, A.(1988) Proc. Natl. Acad. Sci. USA 85, 4051-4055.

28. Gilbert, F., Feder, M., Balaban, G., Brangman, D., Lurie, D. K.,Podolsky, R., Rinaldt, V., Vinikoor, N. & Weisband, J. (1984) CancerRes. 44, 5444-5449.

29. Davis, M., Malcolm, S., Hall, A. & Marshall, C. J. (1983) EMBO J. 2,2281-2283.

30. Francke, U., de Martinville, B., Coussens, L. & Ullrich, A. (1983)Science 222, 1248-1250.

31. Zang, K. D. (1982) Cancer Genet. Cytogenet. 6, 249-274.32. Yamada, K., Kondo, T., Yoshioka, M. & Oami, H. (1980) Cancer Genet.

Cytogenet. 2, 293-307.33. Seizinger, B. R., Martuza, R. L. & Gusella, J. F. (1986) Nature (London)

322, 644-647.34. Whang-Peng, J., Triche, T. J., Miser, J., Douglass, E. C. & Israel, M. A.

(1984) New Engi. J. Med. 311, 584-585.35. Vigfusson, N. V., Allen, L. J., Phillips, J. H., Alshibaja, T. & Riches,

W. G. (1986) Cancer Genet. Cytogenet. 22, 211-218.

Neurobiology: Lieberburg et al.