DNA AND CELL BIOLOGYVolume 15, Number 11, 1996Mary Ann Liebert, Inc.Pp. 965-974

Human Kinesin Light (/3) Chain Gene: DNA Sequence andFunctional Characterization of Its Promoter and First Exon

YUTI CHERNAJOVSKY, ALEX BROWN,1 and JULIE CLARK

ABSTRACT

Kinesins are tubulin molecular motors whose function is to transport organdíes within cells. Very little isknown about the regulation of expression of these proteins. We have characterized the gene product of one

differentially spliced mRNA of the human light chain kinesin and cloned its promoter region. A full-lengthkinesin cDNA was translated in vitro in a cell-free system, producing a 70-kDa protein. Using this cDNA as

a probe, we isolated and sequenced the promoter, first exon, and part of the first intron of this gene from a

genomic A EMBL3 human placental DNA library. The whole gene spans more than 90 kb. The ß kinesin pro-moter region confers only constitutive transcription to the bacterial chloramphenicol acetyltransferase (CAT)reporter gene. In permanently transfected human HeLa and NB100 neuroblastoma cells, a reporter gene con-

taining the promoter and part of the first exon of ß kinesin was 75-fold more active than the HSV-tk pro-moter. The first exon contains the 5'-untranslated sequence capable of forming a stable double-hairpin loop,which functions as a translational enhancer. Its deletion decreases the efficiency of in vitro translation of ßkinesin mRNA and confers increased translation to a CAT reporter gene.

INTRODUCTION

Kinesins are molecular motor proteins implicated in theintracellular movement of organdíes (Brady and Pfister,

1991; Brady 1991; Endow, 1991) and in the movement of chro-mosomes along microtubules during cell division. In sea urchin(Johnson et al., 1990) and mammalian cells (Pfister et al,1989), kinesins have been characterized as tetrameric proteins.Two subunits are the heavy chains (a chains) of a relative mol-ecular mass of approximately 120 kDa and two light chains (ßchains) of approximately 70 kDa (Pfister et al, 1989; Valléeand Shpetner, 1990). Intracellular organdíes move inside thecell along microtubules by attaching to the tetrameric aodßßkinesin molecular motor (Vale and Goldstein, 1990). The a

chains provide the tubulin binding site and the ATPase domain(Yang et al, 1990), whereas the ß chains are responsible forthe specific attachment of the organdie to be moved by the ki-nesin tetramer (Vallée and Shpetner, 1990). Kinesins are in-volved in axonal transport of organelles in brain cells (Brady

and Pfister, 1991) and have been reported to be associated withthe mitotic (Sawin et al, 1992) and meiotic apparatii (Haganand Yanagida, 1992; Theurkauf and Hawley 1992) and inter-phase cytoplasm in sea urchin (Wright et al, 1991). Kinesinstransport their bound organelle to the plus end of the micro-tubule (Schnapp et al, 1992).

At present, three rat brain kinesin ß chain cDNAs have beencloned (Cyr et al, 1991), and a partial cDNA sequence (namedEST00761) of human brain origin has been reported (Adams et

al, 1992), and a full-length human cDNA from T cell originhas been cloned (Cabeza-Arvelaiz et al, 1993). The three ratcDNAs are the product of one gene spliced differentially at the3'-end, producing different COOH terminal ends in the protein.A similar differential splicing occurs in sea urchin (Wedamanet al, 1993). This splicing mechanism seems to confer the ki-nesin specificity for organelle binding.

Here we report the expression in vitro of the T cell-derivedfull-length human ß kinesin cDNA and the functional charac-terization of the promoter and first exon of this gene.

Kennedy Institute of Rheumatology, Molecular Biology Laboratory, Hammersmith, London W6 7DW, UK^Present address: NIMR, The Ridgeway, Mill Hill, London, NW7 1AA, UK.

965

966 CHERNAJOVSKY ET AL.

MATERIALS AND METHODS

Plasmids and human genomic DNA libraryA human kinesin ß chain cDNA (EMBL database accession

number L04733)(Cabeza-Arvelaiz et al., 1993) from a T celllibrary (therefore, named ßi kinesin for originating from an im-mune cell) in pGem3Z was kindly provided by Dr. L.B.Lachman (Department of Cell Biology, M.D. Anderson CancerCenter, Houston, TX). The reporter plasmids pBLCAT-9+(KJein-Hitpass et al., 1988), NSE-CAT (Forss-Peter et al,1990), and pSV2L (de Wet et al, 1987) and the selectable plas-mid pSV2neo (Southern and Berg, 1982) were described pre-viously. The human genomic placental DNA library in ÀEMBL3 phage (Frischauf et al, 1983) was from Clontech.Plasmids were cloned and maintained in Escherichia coli DH5acells (Hanahan, 1983).

In vitro translation of kinesin cRNA

Capped cRNA was synthesized using T7 RNA polymerasefrom a pGEM3z (Promega, Madison, WI) plasmid carrying thefull-length ßi kinesin cDNA. This cRNA was translated in vitrousing a rabbit reticulocyte lysate, with or without a dog pan-creas microsomal membrane preparation (Promega, Madison,WI), using [35S]methionine (Amersham, U.K.) and under theconditions recommended by the supplier. After translation, theproducts were separated on SDS-acrylamide gel (Laemmli,1970). Gel fluorography was performed by treating the gel with1 M sodium salicylate, dried, and exposed to autoradiography.

Screening of human genomic DNA libraryThirty plates (15 cm in diameter) were cultured with 20,000

phages each. After transfer to nylon membranes (Nytran,Schleicher and Schiiell, Keene, NH) and crosslinking with UVlight, the filters were probed with the full-length cDNA (2.4kb) of human kinesin light chain labeled by random priming(Feinberg and Vogelstein, 1984), as recommended by the sup-plier. Salmon sperm and E. coli DNA (at 50 pglml each) were

used during the prehybridization and hybridization steps.

Southern blot hybridizationGenomic DNA was extracted after proteinase K treatment of

whole cell SDS-lysates and spooling as previously described(Chernajovsky et al, 1984). cDNA fragments were labeled byrandom priming (Feinberg and Vogelstein, 1984) using a

[32P]dCTP (6000 Ci/mmol, Amersham) and hybridized to elec-trophoresed nucleic acids transferred (Southern, 1975; Thomas,1980) to nylon membranes (Nytran, Schleicher and Schiiell),crosslinked with UV light. Hybridization was carried out in thepresence of5%dextran sulfate, 5 X SSC, 0.5% fat-free dry milk(Blotto), 0.5% SDS, and 1-5 x 106 cpm/ml of labeled probe.

To build a reporter gene driven by the ß kinesin gene pro-moter, we first removed the HSV-tk promoter from the vectorpBLCAT9+ (Klein-Hitpass et al, 1988), using HindIII andBglll, and blunted these ends with Klenow fragment of DNApolymerase and dNTPs. The HindIII to Asp718 3.6 kb fragmentfrom the kinesin promoter region was similarly blunted and lig-ated to this vector. The orientation of the insert in relation tothe CAT gene was assessed by restriction with BamHI and

Bglll. The plasmid containing the promoter in the right orien-tation regarding the CAT gene was named clone 6.

Restriction enzyme mapping and DNA sequencingDNA was digested with restriction enzymes in single and

double digests, using restriction enzymes from BoehringerManheim (Lewes, UK) according to their recommendations.DNA sequencing by the dideoxy sequence termination method(Sanger et al, 1977) was determined from both strands and per-formed using T7 DNA polymerase (Sequenase, USB,Amersham, UK) after subcloning Smal and Asp718 fragmentsfrom the 4.6 kb Eco Rl-Sal 1 promoter region into pUC18/19(Yanisch-Perron et al, 1985).

Removal of hairpin structure at 5 '-untranslated regionof kinesin cDNA

To remove most of the hairpin structure (positions 133 to255), we digested the cDNA clone at the unique restriction sitesNar 1 and Sau 1 (positions 116 and 240, respectively). The pro-truding ends were filled in with Klenow fragment of DNA poly-merase and dNTPs and relegated with T4 DNA ligase. Then,produced capped cRNA from the T7 promoter was translatedinto rabbit reticulocyte lysate (Promega, Madison, WI). Usingdilutions of the cRNA, we compared the efficiency of transla-tion to the original cDNA (+HP) to the cDNA lacking the hair-pin structure (-HP). The cell free system translated a unique65 kDa protein not present without cRNA and that correspondsvery well with the expected 66 kDa protein deduced from theopen reading frame (ORF) of the cDNA. The [35S]methionine-labeled translation product was quantitated after SDS-PAGEwith an Instant Imager (Packard, Meridine, CT).

Addition of hairpin structure at 5 '-untranslated regionto a heterologous gene

We cloned the same hairpin fragment Nar 1-Sau 1 into theBglll site located 50 nucleotides downstream of the transcrip-tion initiation site of the HSV-tk in the pBLCAT-9 reporterplasmid. To assess its effect on translation efficiency, we tran-siently transfected COS cells with increasing amounts of DNAfrom the CAT-containing plasmids (5-20 pg) with a constantamount (5 pg) of the pSV2L firefly luciferase expression plas-mid (de Wet et al., 1987) and decreasing amounts of pUC18DNA (Yanisch-Perron et al, 1985) to bring about a final 25 ¿igDNA per transfection.

Cell culture selection of permanently transfected HeLaand NB100 cells

HeLa, COS-7, and NB100 neuroblastoma cells were grownin DMEM, 10% fetal bovine serum (FBS) supplemented with100 U/ml penicillin and 100 pg/ml streptomycin at 37°C with10% CO2 in a humidified atmosphere incubator. HeLa cells wereobtained from ICRF (London, UK), and the human neuroblas-toma MB 100 cell line was kindly provided by Dr. A. Evans,Children's Hospital Philadelphia, PA). Cells were transfected bythe calcium phosphate coprecipitation method (Chernajovskyand Kirby-Sanders, 1990) with 20 pg of reporter plasmid and 1pg of selectable plasmid. After transfection, cells were treatedwith 10% glycerol in DMEM for 4 min, washed twice with

HUMAN KINESIN ß CHAIN GENE 967

DMEM serum-free medium, allowed to recover for 48 hr inDMEM with 10% FBS, and then trypsinized and diluted 1:4into medium containing 1 mg/ml G418. After 2-3 weeks,30^10 clones were pooled and grown as a single populationof cells. CAT assays were performed as described previously(Chernajovsky and Kirby-Sanders, 1990; Gorman et al, 1982).The concentrations of the stimulators used (PMA, forskolin, andcholera toxin) have been used previously by us and by others(Jameson et al, 1986; Ray et al, 1988; Chernajovsky and Reid,1990).

RESULTS

Isolation of the ß kinesin geneSouthern blot analysis of human DNA with a full-length ß

kinesin cDNA probe indicated that this gene is large. TheHindlll restriction fragments that hybridized to the kinesincDNA probe were of 7.5, 5.8, 5.4, 5.0, 4.8, 3.2, 2.1, 1.9, 1.8,

Table 1. Summary of Different A EMBL-3 PhageIsolates Hybridizing to ß Kinesin cDNA Fragments"

Eco Rl Xho 1Phage fragments fragments 5 '-probe0 3 '-probe2.1B

211.B/211.A

182183

52

291

41

262

21A

161

21.413.84.34.1

18155.24.3

452517

45

23222518

3014

45

1817103.8

18179.4

209.54.22.3

23189.54.44.2

2520232222159.54.2

21189.7

219.79.56

17129.35

Yes No

No Yes

Weakly No

Weakly Weakly

No Weakly

Weakly No

No Yes

No Weakly

Yes No

Yes No

"Phage DNA was digested with the restriction enzyme indi-cated and hybridized either to a 0.3 kb Hindlll 5'-probe or withthe remaining 2 kb 3'-end of the cDNA.

b5'-End probe is a 0.3 kb Hindlll fragment of cDNA, 3'-endprobe is the 2 kb of remaining cDNA sequence.

r <?&*r^

12.28.17.16.15.0 _

4.0 —

3.0 —

12.0

1.6

Bphage 2. t B

CÄ19 4

\plasmid E/S \

VTi i r TT

FIG. 1. (A) Southern blot of HeLa cell DNA. Genomic DNA(15 pg) was restricted overnight with EcoRl or Hindlll and an-

alyzed by Southern blotting with random primed ß kinesincDNA (see Materials and Methods). (B) Restriction enzyme mapof phage 2. IB and 4.6 kb of promoter region. DNA from phage2. IB was digested with several restriction enzymes. The posi-tions of the Eco Rl sites, vector Sal 1 sites, À EMBL3 arms andcohesive ends (COS) are indicated. The 4.6 kb fragment Sal1-Eco Rl was subcloned into pUC 19 (plasmid E/S), and fur-ther restriction enzyme mapped. The arrow indicates the direc-tion of transcription and the area that has been sequenced. Notethe different scales on the phage 2.IB and the plasmid E/S maps.

and 1 kb, whose sun is 38.5 kb (Fig. la). To study the mecha-nisms of gene expression of ß kinesin, we isolated its gene. Tothis end, we screened a human placental genomic DNA librarycloned in the A EMBL3 phage vector. In the first screen, 33

968 CHERNAJOVSKY ET AL.

-249CCTGCCCCGA GAGCCCCACC CCCGTCCGCG TTACAACCGG AAGGCCGCTG

-199GGTCCTGCAC CGTCACCCTC CTCCCTGTGA CCGCCCACCT GACACCCAAA

-149CAACTTTCTC GCCCCTCCAG TCCCCAGCTC GCCGAGCGCT TGCGGGGAGC

CffiGCCGGCG CGCGGCATTG TG

CACCCAGCCT CAGTTTCCCC AGCCCCG^G&XSGGGCGAGGG GCGATGACGTIS y y s y y y ,

Sma I

99

-49CGCCGGGGGG

AGCAACACTG AGACGCCATT TCJGGCGGCCG GGACGGGCGC AAGGCGGG

+1

AGCGGGACTG GCTGGGTCGG CTGGGCTGCT GGTGGAGGAG CCGCGGGGC2?2102

GTGCTCGGCG GCCAAGGGGA CAGCGCGTGG GTGGCCGAGG ATGCTGCGGG

152GCGGTAGCTC CGGCGCCCCT CGCTGGTGAC TGCTGCGCCG TGCCTCACAC

AGCCGAGGCG GGCTCGGCGC ACAGTCGCTG CTCCGCGCGC GCGCCCGGC>G202

GCGCTCCAGG TGCTGACAGC GCGAGAGAGC GCGGCCCTCA GGAGCAAGGC252

GGTGAGTCCC CGCGTCGTCG CCCCGGACCG CGGCCCCCTC CTCATCCTCC302

352GCCCCGTCCC TGTCCCGCTC CTCTTCGGAC CCGCCCCGGC CGCAACTCTG

TCCCCATCCA GGCCTCCTTC CCGGTTTGGT CCCGGCCCCT CTCCGTTCCC.402

452ACCCCGGTAC CCGCCCCAGT TCACCGCCCC GGCCGGTCCG CGACCCCTTC

Asp 718 502TAGGTTCAGG TCGGGTTCTT GTCCCCGGCC CTTTTGCCAG CCCCGGCTCC

CGGCGCCGCG CGTCCTCCCC ATCCGCGTCC CACTGCAG'Pst

,540

f~~l TATA box SP1 X///A CREB

FIG. 2. DNA sequence of the 5'-flanking and first exon/intron of the ß kinesin gene. DNA sequence (838 nucleotides) of the5'-flanking, exon, and intron of the ß kinesin gene. Numbers indicate position of the nucleotide after the initiation site of tran-scription (+1) or upstream of it (negative numbers). The first exon is in italics and underlined. Restriction enzyme sites are un-derlined and indicated. Putative TATA box, SP1, and CREB binding sites are in shaded areas. This sequence has been sent tothe EMBL nucleotide sequence database, and Accession number X69658.

plaques were isolated, from which 11 were true positives andreached the third screening and purification step. These clonescould be separated into 10 groups according to their restrictionpattern. Table 1 shows a summary of the phage clones obtained

and grouped by their Eco Rl and Xho 1 restriction patterns. Therestriction data presented have been kept to a minimum.

We have also restricted these phage clones with HindIII,Xbal, Bgl II, Smal, PstI, and double digests of Eco R 1/Xbal,

HUMAN KINESIN ß CHAIN GENE 969

82 Met Leu Arg Gly Gly Ser Ser Gly Ala Pro Ser Trp stophuman GGGTGGCCGAGG ATG CTG CGG GGC GGT AGC TCC GGC GCC CCT CGC TGG TGArat GTCGGGGTGAGG ATG CTG CGG GGC GGC TGC GGA GGC GTC GCT TGC TGC TGA

Met Leu Arg Gly Gly Cys Gly Gly Val Ala Cys Cys stop3

142 257Met Tyr

human CTGCTGCGC.ATG TAT.rat GGCGGCTGG.ATG CAT.

Met His62 121

FIG. 3. Homologous short open reading frame in the 5'-untranslated regions of human and rat ß kinesin mRNAs. Partial DNAsequence of the 5'-untranslated region of both human and rat ß kinesin mRNAs is shown. The putative short open reading framesare shown above (human) and below (rat) the DNA sequence. Numbers correspond to the position of the RNA start in the hu-man mRNA and to the first nucleotide of the cDNA reported by Cyr et al, 1991. Cytosine-123 in the human sequence (under-lined) appears as an adenosine in the human cDNA clone, probably indicating an allelic difference. The second ATGs indicatedcorrespond to the longest open reading frames on the cDNAs.

Hindlll/Pstl, Hind III/ Bgl II, and Hindlll /Smal. The patternof hybridizing bands was quite varied, and thus we concludedthat these clones were not overlapping and corresponded to dif-ferent portions of the gene. We estimated the minimum size as

90 kb because in the 5' phage containing the promoter region,9 kb is the size of the intron sequence. We have isolated 10 dif-ferent phages (9 kb X 10 = 90). These results indicate that theintrons of the kinesin gene are large. We decided to concen-

trate on the 5'-flanking region of the kinesin gene. To isolatethe promoter region, we used the 5' Hindlll fragment of thecDNA (0.3 kb) as a probe and further characterized clone 2. IB.The restriction map of clone 2. IB is shown in Figure lb. The4.6 Eco Rl-Sal 1 fragment was further subcloned into pUC19.Smaller fragments that hybridized with the 0.3 kb 5'-end of thecDNA were also subcloned (Pst 1-1.2 kb and Pst l-Smal-0.6kb). The full DNA sequence of the Pst 1-Sma I (0.6 kb) frag-ment and additional sequence of the 5'-genomic flanking se-

quence is shown in Figure 3. These sequences encompass thepromoter area, first exon together with part of the first intronof the ß kinesin gene. This intron has at least 9 kb in length as

it extends to the end of the 2.IB phage.The promoter sequence contains several putative transcrip-

tion factor binding sites, including SP1, CREB, and TATAbox (Fig. 2). The TATA box sequence, although unconven-

tional, is located as expected at 30 nucleotides upstream ofthe start of the characterized cDNA clone (Cabeza-Arvelaizet al, 1993). This nonconsensus TATA box (Corden et al,1980), located at —29 to —34, contains a cytosine as first nu-

cleotide, as do the human globin genes (Proudfoot et al,1980). This TATA box could be functional, as TF II is knownto bind to nonconsensus sequences, and its interaction withDNA may be modulated by other transcription factors (Hahnet al, 1989).

The first exon is 253 nucleotides long and corresponds to the5'-untranslated region of the mRNA. Comparison of this DNAregion with cloned rat cDNAs showed, as expected, evolution-ary divergence at this area but, surprisingly, also showed ho-mology in an area with potential for encoding a small peptide,12 amino acids long in humans and 12 amino acids long in ratkinesin light chain A and C—that is, not preceded by a con-

A GC C'A C'

c-g'U-AC-GC-G

/^GU G\ U c'

G-CC-GC-GG-CC-GG-CU-A

< IG-CU-AC-G

U-AC-GG-C

V*A

U-A'G-CC-GU-AC-GC-G

A

c CGC CGCGv G C G-GCGCUG-C'

/UCC-CGA G G G C

C-C' V UG

k yAG,CGCG AGC G C GA VG A'C

FIG. 4. Potential double hairpin structure at the 5'-untrans-lated region of human ß kinesin mRNA. The sequence of themRNA from nucleotides 133-255 was analyzed using theRNAFOLD program contained in the University of WisconsinGenetics Computer Group (GCG) package at the HumanGenome Mapping Project Resource Centré, Harrow, London

970 CHERNAJOVSKY ET AL.

A. NB100 cells

S S

£82 oo i:

pBLCAT9+NSE CATClone 6

Control Forskolin

B. Hela cells

> r-N

<CJ3Ío £

pBLCAT94Clone 6

Control PMA Cholera toxin

Treatment

Forskolin

FIG. 5. ß Kinesin promoter constitutively drives trancription of a reporter gene. HeLa or NB100 cell extracts of 1 X 106 per-manently transfected cells with clone 6 plasmid (0.2 pg protein/assay) or the control plasmids pBLCAT9+ (15 pg protein/as-say) were analyzed for their CAT activity (see Materials and Methods). Cells were untreated (control) or treated overnight withcholera toxin (250 ng/ml), forskolin (50 pM), or phorbol myristate acetate (PMA) at 0.2 pglml. The result of one of three sim-ilar experiments is shown.

sensus ribosomal binding site (Kozak, 1986) (Fig. 3). This cod-ing region is followed by a GC-rich region capable of forminga very stable secondary mRNA structure (AG= —52.2kCal/mol) (Fig. 4). Interestingly, only the human mRNA couldform a double hairpin structure, which requires 123 nucleotides.The rat mRNA has only 67 nucleotides in this region, beforethe kinesin protein start site, and its potential single hairpinstructure has lower stability (AG= —23.8 kcal/mol).

Functional characterization of the promoter regionTo show that the promoter region that we have cloned and se-

quenced is functional, we constructed a reporter plasmid in whichthe ß kinesin promoter drives transcription of the bacterial chlo-ramphenicol acetyltransferase gene and named it clone 6. We co-transfected clone 6 with the selectable marker pSV2neo intoHeLa cells. As a control, we cotransfected the plasmidpBLCAT9+, which has the HSV thymidine kinase (HSV-tk)

promoter driving the same bacterial reporter gene, with pSV2neo.Similarly, we cotransfected clone 6, pBLCAT9+, and the neu-

ron-specific enolase promoter (NSE)-driven chloramphenicolacetyltransferase into human NB100 neuroblastoma cells. Stablepopulations of transfected cells of more than 30 clones were se-lected in G418, grown, and analyzed for their CAT expression.

As expected from the steady-state levels of kinesin mRNAin several cell lines (data not shown), the expression from thekinesin promoter in the reporter gene plasmid was also consti-tutive, as was the expression from the HSV-tk and NSE pro-moters in the control reporter plasmids. Calibration of theamount of protein extract necessary to obtain a linear CAT as-

say showed that cells transfected with clone 6 had 75-fold to100-fold more CAT activity per microgram of protein extractthan extracts from pBLCAT9+ transfected cells (Fig. 5). Inother parallel experiments of transient expression in COS-7cells, clone 6 was as active as a CMV promoter and better thanan SV40 early promoter (unpublished observations).

HUMAN KINESIN ß CHAIN GENE 971

12 3 4 5 6

97.4

69

45

30

FIG. 6. In vitro translation of cRNA for ß kinesin. A ß ki-nesin cDNA cloned into the plasmid pGEM3z was linearizedat the unique Apa /site located 3 ' of the translational stop codonand transcribed to cRNA with T7 RNA polymerase in the pres-ence of the cap analog 7-methyl-GpppG. The cRNA was trans-lated in vitro in rabbit reticulocyte lysate with or without dogpancreas microsomal membranes (+ or -). As controls, thecRNAs for ß lactamase (signal peptide processed; therefore,mobility increases, and in this gel it run out) and a factor (thatis, glycosylated, and its molecular weight increases as a resultof glycosylation) are shown. The samples were treated with 5%mercaptoethanol and loaded onto a 10% SDS-Page. Lanes 1and 2, ß kinesin; lanes 3 and 5, a factor; lanes 5 and 6, ß lac-tamase. The positions of the relative molecular mass markersare indicated at left.

D.

+HP

-HP

100 150 200

cRNA (ng/reaction)

-O- pBLCAT-9_«_pBLCAT-9 + HP

CAT DNA ( ng /transfection)

FIG. 7. The hairpin at the 5'-untranslated region of ß kinesinmRNA is necessary for efficient translation. (A) Cell free trans-lation of kinesin cDNA with or without the hairpin. cRNA froma plasmid with or without the hairpin (-HP) was transcribedafter Apa 1 linearization and translated in a reticulocyte lysateas before (Fig. 6). The results after scanning the 70 kDa kinesintranslated peptide were plotted against the cRNA concentration.This experiment was repeated three times with similar results.(B) Transient reporter gene expression with or without the hair-pin structure. Increasing amounts of pBLCAT9 with (+) or

without the hairpin were transiently transfected into COS-7cells. CAT activity is plotted against quantity of DNA after cor-

recting for transfection efficiency using a constant amount ofpSV2L DNA in each sample (see Materials and Methods).

Attempts were made to upregulate the kinesin promoter withsecond messenger analogs of the cAMP kinase signaling path-way or protein kinase C. Cholera toxin, forskolin, and PMAfailed to induce substantially expression of the reporter genes.On the contrary, they were slightly inhibitory to the ß kinesinpromoter and strongly inhibitory for the control HSV-tk pro-moter driven plasmid in HeLa cells (Fig. 5). In NB100 cells,they were slightly stimulatory. In similar experiments, a reporterluciferase gene with the human IL-6 promoter was induced 30-fold by PMA (data not shown). No substantial differences were

obtained with the kinesin promoter in the presence of these sec-

ond messenger analogs in either cell line that was not mirroredby the control reporter plasmids used. These results confirm theNorthern blot data obtained in human lung fibroblasts treatedwith second messenger analogs that did not alter ß kinesinmRNA levels (unpublished) observations.

In vitro translated ß kinesin is not posttranslationallymodified by dog pancreas microsomal membranes

The longest open reading frame of kinesin has a putative Asnglycosylation site (sequence NMS, amino acids positions 4-6).To test whether this glycosylation site was used, we translatedits capped cRNA in a rabbit reticulocyte lysate in the presenceor absence of dog pancreas microsomal membranes. Whereas thecontrol mRNAs for a factor and ampicillinase were glycosylated(Fig. 6, lanes 3 and 5) or cleaved, respectively, the 70 kDa ß ki-nesin protein was not modified (Fig. 6, lane 1).

Removal of hairpin structure at 5 '-untranslated regiondecreases translation efficiency of ß-kinesin mRNA

To test for the function of the stable hairpin structure (Fig.4), we removed it from the cDNA clone by using restriction di-

972 CHERNAJOVSKY ET AL.

gestion and then produced capped cRNA from the T7 promoter.Using dilutions of the cRNA, we compared the efficiency oftranslation to the original cDNA in the rabbit reticulocyte cellfree system. Figure 7A shows that the cRNA without the hair-pin-loop structure is less efficient in translation than the origi-nal cDNA, which showed higher translation rates at low mRNAconcentrations.

Addition of hairpin structure at 5 '-untranslated regionof heterologous gene increases translation efficiency

We also tested the reverse situation by cloning the hairpinstructure in the 5'-untranslated region of a heterologous gene.For this purpose, we cloned the same hairpin fragment Nar1-Sau 1 into the Bgl II site located 50 nucleotides downstreamof the transcription initiation site of the HSV-tk in the pBLCAT-9 reporter plasmid. To assess its effect on translation efficiency,we transiently transfected COS cells with increasing amountsof DNA from the CAT containing plasmids (5-20 pg) with a

constant amount (5 pg) of the pSV2L firefly luciferase ex-

pression plasmid and decreasing amounts of pUC 18 DNA tobring about a final 25 pg DNA per transfection. Figure 7Bshows that the hairpin fragment of kinesin increases the trans-lation efficiency 4-fold to 5-fold when compared withpBLCAT-9 lacking this sequence.

DISCUSSION

The finding in the rat (Cyr et al, 1991) and more recently insea urchin (Wedaman et al., 1993) that ß kinesin cDNAs are dif-ferentially spliced at the 3'-end of the mRNA, producing kinesinshaving different COOH ends, indicates that postranscriptionalregulation of this gene may be very important for its various func-tions. This finding also indicates that to study ß kinesin func-tion^), each of the differentially spliced mRNAs will need to bestudied as separate entities. For this purpose, we undertook theexpression of a full-length human ß kinesin cDNA in vitro. Wehave shown that this ß kinesin cDNA encodes a 70 kDa protein,which could not be modified by microsomal membranes despitethe fact that it contained an Asn glycosylation site at its amino-terminal end. The lack of a clear transmembrane signal sequencemay exclude this protein from processing in microsomes.

This work also shows the sequence of the promoter regionand first exon of the human ß kinesin gene. The promoter re-

gion transcribes constitutively, as demonstrated by the consti-tutive expression of the CAT bacterial reporter gene and alsobe the steady-state levels of ß kinesin mRNA in a variety ofcells. Although the promoter region contains DNA binding sitesfor cAMP-modulated transcription factor (CREB), it does notseem to play a strong regulatory role in the established cell linestested, as only a slight modulatory effect was observed usingforskolin or cholera toxin. It is possible, however, that this ex-acting element is used during development, differentiation, or

transformation processes.The first exon of this gene contains the 5'-untranslated re-

gion of the ß kinesin mRNA. As found in other eukaryoticmRNAs, such as mRNAs for cytokines and oncogenes (Kozak,1986), the ß kinesin mRNA contains a short open reading framethat is conserved between rat and human. This open reading

frame is different from the one reported in the kinesin heavychain cDNA (Navone et al., 1992) and does not contain up-stream a consensus ribosomal binding site. It is thought thatsuch short open reading frame is advantageous to help the ri-bosome slide to a stronger internal consensus ribosomal site be-cause the first AUG codon lies in an unfavorable context forinitiation (Kozak, 1986). In our case, the lack of consensus ri-bosomal binding site may prevent translation of this short openreading frame sequence. However, its conservation in evolu-tion is intriguing.

Another salient feature of the first exon is its capability offorming a stable double hairpin structure. Such a structure couldbind translational repressors (Waiden et al., 1988) or enhancers(Pelletier and Sonenberg, 1988) or serve as a binding site forother regulatory molecules important during embryogenesis ordifferentiation. Deletion of this hairpin structure showed re-duction in the translation efficiency in vitro, indicating the im-portance of this sequence. It could be speculated that the roleof this hairpin structure is to compensate for the dilution of a

specific mRNA caused by splicing, and by ensuring efficienttranslation of all spliced ß kinesin mRNAs, the concentrationof these proteins in the cell are maintained at a sufficient level.The fact that this sequence could confer increased translationto the CAT gene indicates that it has a role in translation. Themechanism by which this hairpin structure increases translationis unknown at present. To determine whether it binds transla-tion factors or increases RNA stability will require further in-vestigation.

Our finding that in a mixed population of permanently trans-fected clones the CAT activity from the ß kinesin promoter is75-fold to 100-fold higher than that from the HSV-tk or NSEpromoters may be reflecting the strength of the kinesin pro-moter or indicating that this double hairpin structure enhancesmRNA translatability. The rat ß kinesin mRNAs have a muchshorter 5'-untranslated region, suggesting that some of this se-

quence is not essential for regulation or that rat and human ßkinesin expression is regulated by different mechanisms. It willbe interesting to know if the shorter 5'-region in the rat mRNAresults from splicing out of sequences from the gene.

The gene structure of the ß kinesin gene is complex and hasa very large introns, as depicted by the fact that 11 phages con-

tain ß kinesin sequences, and the first exon alone (254 nu-

cleotides) is followed by more than 9 kb of sequences (mostlyintron sequences) incapable of hybridization with cDNA inphage 2.IB. A more accurate assessment of the length of thisgene could be obtained from mapping cosmid or YAC genomicclones.

From all the features of the gene and its product describedhere, it appears that ß kinesin expression is mainly regulated atthe posttranscriptional and translational levels.

ACKNOWLEDGMENTS

We thank Dr. I. J. Fidler for his support during the first stagesof these studies, Dr. Subramani for the pSV2L plasmid, Drs.M. Feldmann, I. Kerr, and R. Fellowes for critical review ofthe manuscript, Mr. D. Bartos, Mrs. H. Kirby-Sanders, and MissA. Hales for technical help at different points on this project,and the computer facilities of the Human Genome Mapping

HUMAN KINESIN ß CHAIN GENE 973

Project Resource Centre, Harrow, London, that greatly facili-tated the handling and analysis of DNA and RNA sequences.

REFERENCES

ADAMS, M.D., DUBNICK, M., KERLAVAGE, A.R., MORENO, R„KELLEY, J.M., UTERBACK, T.R., NAGLE, J.W., FIELDS, C, andVENTER, J.C. (1992). Sequence identification of 2,375 human braingenes. Nature 355, 632-634.

BRADY, S.T. (1991). Molecular motors in the nervous system. Neuron7, 521-533.

BRADY, ST., and PFISTER, K.K. (1991). Kinesin interactions withmembrane bounded organdíes in vivo and in vitro. J. Cell. Sei.14(Suppl.), 103-108.

CABEZA-ARVELAIZ, Y., SHTH, L.C.N., HARDMAN, N., ASSEL-BERGS, F., BILBE, G., SCHMITZ, A., WHITE, B., SICILIANO,M.J., and LACHMAN, L.B. (1993). Cloning and genetic character-ization of the human kinesin light-chain (KLC) gene. DNA Cell Biol.12, 881-892.

CHERNAJOVSKY, Y., CHEN, L., MARKS, Z., NOVICK, D., RU-BINSTEIN, M., and REVEL, M. (1984). Efficient constitutive pro-duction of human fibroblast interferon by hamster cells transformedwith the IFN-/31 gene fused to an SV40 early promoter. DNA 3,297-308.

CHERNAJOVSKY, Y., and KIRBY-SANDERS, H.M. (1990). A ex-

acting sequence, located at -450 in the promoter of the human in-terferon-inducible gene 6-16, binds constitutively to a nuclear pro-tein and decreases the expression of a reporter interferon-induciblepromoter. Lymphokine Res. 9, 199-212.

CHERNAJOVSKY, Y., and REÍD, T.R. (1990). Regulation of the hu-man interferon-inducible 6-16 promoter in tumor necrosis-sensitiveand resistant mouse cells: cAMP as a mediator of signal transduc-tion. J. Interferon Res. 10, 627-636.

CORDEN, J., WASYLYK, B., BUCHWALDER, A., SASSONI-CORSI, P., KEDINGER, C, and CHAMBÓN, P. (1980). Expressionof cloned genes in a new environment. Science 209, 1406-1414.

CYR, J.L., PFISTER, K.K., BLOOM, G.S., SLAUGHTER, CA., andBRADY, S.T. (1991). Molecular genetics of kinesin light chains:generation of isoforms by alternative splicing. Proc. Nati. Acad. Sei.USA 88, 10114-10118.

de WET, J.R., WOOD, K.U., DELUCA, M., HELINSKI, D.R., andSUBRAMANI, S. (1987). Firefly luciferase gene: structure and ex-

pression in mammalian cells. Mol. Cell. Biol. 7, 725-737.ENDOW, S.A. (1991). The emerging kinesin family of microtubule

motor proteins. Trends Biochem. Sei. 16, 221-225.FEINBERG, A.P., and VOGELSTEIN, B. (1984). A technique for ra-

diolabelling DNA restriction endonuclease fragments to high spe-cific activity. Anal. Biochem. 132, 6-13.

FORSS-PETER, S., DANIELSON, P.E., CATSICAS, C, BATTEN-BERG, E., PRICE, J., and SUTCLIFF, G. (1990). Transgenic miceexpressing beta-galactosidase immature neurons under neuron-spe-cific enolase promoter control. Neuron 5, 187-197.

FRISCHAUF, A.-M., LEHRACH, H., POUSTKA, A., and MURRAY,N. (1983). Lambda replacement vectors carrying polylinker se-

quences. J. Mol. Biol. 170, 827-842.GORMAN, CM., MOFFAT, L.F., and HOWARD, B.H. (1982).

Recombinant genomes which express chloramphenicol acetyltrans-ferase in mammalian cells. Mol. Cell. Biol. 2, 1044—1051.

HAGAN, I., and YANAGIDA, M. (1992). Kinesin-related cut7 proteinassociates with mitotic and meiotic spindles in fission yeast. Nature356, 74-76.

HAHN, S., BURATOWSKI, S., SHARP, P.A., and GUARENTE, L.(1989). Yeast TATA-binding protein binds to TATA elements withboth consensus and non-consensus DNA sequences. Proc. Nati.Acad. Sei. USA 86, 5718-5722.

HANAHAN, D. (1983). Studies on transformation of E. coli with plas-mid. J. Mol. Biol. 166, 557-565.

JAMESON, J.L., JAFFE, R.C, GLEASON, S.L., and HABENER, J.F.(1986). Transcriptional regulation of chorionic gonadotropin a andß subunit gene expression by 8-bromo-adenosine 3',5'-monophos-phate. Endocrinology 119, 2560-2567.

JOHNSON, C.S., BUSTER, D., and SCHOLEY, J.M. (1990). Lightchains of sea urchin kinesin identified by immunoadsorption. CellMotil. Cytoskeleton 16, 204-213.

KLEIN-HITPASS, L., RYFFEL, G.U., HEITLINGER, E., and CATO,A.C.B. (1988). A 13bp palindrome is a functional estrogen respon-sive element and interacts specifically with estrogen receptor.Nucleic Acids Res. 16, 647-663.

KOZAK, M. (1986). Bifunctional mRNAs in eukaryotes. Cell 47,481^183.

LAEMMLI, U.K. (1970). Cleavage of structural proteins during the as-

sembly of the head of bacteriophage T4. Nature 227, 680-685.NAVONE, F., NICLAS, J., HOM-BOOHER, N., SPARKS, L., BERN-

STEIN, H.D., McCAFFREY, G., and VALE, R.D. (1992). Cloningand expression of a human kinesin heavy chain gene: interaction ofthe COOH-terminal domain with cytoplasmic microtubules in trans-fected CV-1 cells. J. Cell Biol. 117, 1263-1275.

PELLETIER, J., and SONENBERG, N. (1988). Internal initiation oftranslation of eukaryotic mRNA directed by a sequence derived frompoliovirus RNA. Nature 334, 320-325.

PFISTER, K.K., WAGNER, M.C, STENOIEN, D.L., BRADY, S.T.,and BLOOM, G.S. (1989). Monoclonal antibodies to kinesin heavyand light chains stain vesicle-like structures but not microtubules incultured cells. J. Cell Biol. 108, 1453-1463.

PROUDFOOT, N.J., SHANDER, M.H.M., MANLEY, J.L., GEFTER,ML., and MANIATIS, T. (1980). Structure and in vitro transcrip-tion of human globin genes. Science 209, 1329-1334.

RAY, A., TATTER, ST., MAY, L.T., and SEHGAL, P.B. (1988).Activation of the human ß2 interferon/hepatocyte-stimulating fac-tor/interleukin 6 promoter by cytokines, viruses and second messen-

ger agonists. Proc. Nati. Acad. Sei. USA 85, 6701-6705.SANGER, F., NICKLEN, S., and COULSON, A.R. (1977). DNA se-

quencing with chain-terminating inhibitors. Proc. Nati. Acad. Sei.USA 74, 5463-5466.

SAWIN, K.E., MITCHISON, T.J., and WORDEMAN, L.G. (1992).Evidence for kinesin-related proteins in the mitotic apparatus usingpeptide antibodies. J. Cell Sei. 101, 303-313.

SCHNAPP, B.J., REESE, T.S., and BECHTOLD, R. (1992). Kinesinis bound with high affinity to squid axon organdíes that move to theplus-end of microtubules. J. Cell Biol. 119, 389-399.

SOUTHERN, E. (1975). Detection of specific sequences among DNAfragments separated by gel electrophoresis. J. Mol. Biol. 98,503-517.

SOUTHERN, P.J., and BERG, P. (1982). Transformation of mam-

malian cells to antibiotic resistance with a bacterial gene under con-

trol of the SV40 early region promoter. J. Mol. Applied Genet. 1,327-341.

THEURKAUF, W.E., and HAWLEY, R.S. (1992). Meiotic spindle as-

sembly in Drosophila females: behavior of nonexchange chromo-somes and the effects of mutations in the nod kinesin-like protein. J.Cell. Biol. 116, 1167-1180.

THOMAS, P.S. (1980). Hybridization of denatured RNA and smallDNA fragments transferred to nitrocellulose. Proc. Nati. Acad. Sei.USA 77, 5201-5205.

VALE, R.D., and GOLDSTEIN, L.S. (1990). One motor, many tails:an expanding repertoire of force-generating enzymes. Cell 60,883-885.

VALLEE, R.B., and SHPETNER, H.S. (1990). Motor proteins of cy-toplasmic microtubules. Annu. Rev. Biochem. 59, 909-932.

WALDEN, W.E., DANIELS-McQUEEN, S., BROWN, P.H.,GAFFIELD, L., RUSSELL, D.A., BIELSER, D., BAILEY, L.C, and

974 CHERNAJOVSKY ET AL.

THACH, R.E. (1988). Translational repression in eukaryotes: partialpurification and characterization of a repressor of ferritin mRNAtranslation. Proc. Nati. Acad. Sei. USA 85, 9503-9507.

WEDAMAN, K.P., KNIGHT, A.E., KENDRICK-JONES, J., andSCHOLEY, J.M. (1993). Sequences of sea urchin kinesin light chainisoforms. J. Mol. Biol. 331, 155-158.

WRIGHT, B.D., HENSON, J.H., WEDAMAN, K.P., WILLY, P.J.,MORAND, J.N., and SCHOLEY, J.M. (1991). Subcellular localiza-tion and sequence of sea urchin kinesin heavy chain: evidence for itsassociation with membranes in the mitotic apparatus and interphasecytoplasm. J. Cell. Biol. 113, 817-833.

YANG, J.T., SAXTON, W.M., STEWART, R.J., RAFF, E.C., andGOLDSTEIN, L.S. (1990). Evidence that the head of kinesin is suf-ficient for force generation and motility in vitro. Science 249,42^17.

YANISCH-PERRON, C, VIEIRA, J., and MESSING, J. (1985).Improved M13 phage cloning vectors and hosts strains-nucleotidesequences of the M13MP18 and PUC19 vectors. Gene 33, 103-119.

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Received for publication March 15, 1996; accepted April 19,1996.