Vol.
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3, 1991
BIOCHEMICAL
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Pages
1991
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PRIMARY STRUCTURE OF MOUSE TYROSINE HYDROXYLASE DEDUCED FROM ITS cDNA Shinichi Ichikawa, Toshikuni Sasaoka,and ToshiharuNagatsu* Departmentof Biochemistry, Nagoya University School of Medicine, Nagoya 466, Japan Received
April
12,
1991
SUMMARY: The cDNAs for tyrosine hydroxylase were cloned from a mousebrain cDNA library by plaquehybridization. Sincethe IongestcDNA clone lacked approximately 150 bp sequenceof its N-terminal region, additional 5’ region was obtainedusingpolymerasechainreaction. Nucleotidesequencedeterminationof cDNAs revealedthat mousetyrosine hydroxylase m-RNA encodes498 amino acidswith a calculatedmolecularmassof 55990. The aminoacid sequenceof mousetyrosine hydroxylase is highly homologusto rat ( 97% ) andhuman( 92% ) enzymes. o 1991 Academic
Press,
Inc.
Tyrosine hydroxylase ( tyrosine 3-monooxygenase;tyrosine, tetrahydropteridine: oxygen oxidoreductase( 3-hydroxylating ), EC l-14.16.2.) ( TH ) catalyzes the fist and rate-limiting stepin catecholaminebiosynthesis( 1 ). This enzyme is expressedin catecholaminergicneuronsin the discreteregionsof the brain, noradrenergicneuronsof sympatheticgangliaand sympatheticnerves,and noradrenalineand adrenalinecells of adrenalmedulla. TH plays a central role in neuronaltransmissionand hormonalaction of catecholamines( 2 ). The cDNA cloning of TH for human ( 3,4,5 ), rat ( 6 ), bovine ( 7 ), quail ( 8 ), and fruit fly ( 9 ) have already beenreported. On the other hand,a laboratory mouseis one of the mostimportant animalsasa standardsystem, and the study of the mousegeneticsbecomeincreasinglyimportant by the advent of transgenicbiology. Recently, transgenicmice over-expressinghumanTH hasbeenproduced ( 10 ). However, mouseTH hasnot beenstudiedintensively. In this communication,we report on the cDNA cloning of mouseTH and the determinationof its nucleotidesequence.
MATERIALS
AND METHODS
Library screening: A mousebrain cDNA library ( strain ICR, 7 weeksold ) in hgt 10 vector was a generousgift from Dr. ShigeoOhno, Tokyo Metropolitan Institute of Medical Science. A humanTH cDNA ( type 4 ) waslabeledwith [a-P32ldCTP ( 3000 Ci / m mol ) by Multiprime DNA labelling system( Amex-sham) and usedas a probe ( 11 * To whom all correspondenceshouldbe addressed. 0006-291X/91 Copyright All rights
$1.50
0 1991 by Academic Press, of reproduction in any form
OK. reserved.
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). A total of 106 k phage plaques on E.&i strain C6CO h~7 lawn cells were screened. The plaques were Lifted onto nylon membranes ( Hybond-N, Amersham ). The membranes were prehybridized for lhr at 42°C in a buffer containing 6 x SSC ( 1 x SSC is 0.15 M NaCl and 0.015 M sodium citrate ), 0.1% SDS, 5 x Denhartdt’s solution, 50% formamide, and salmon sperm DNA (100 pg./ ml ). Hybridization followed at 42°C for 24 hrs in the above specified buffer with the labeled human TH cDNA ( type 4 ). The membranes were washed at 42” C with 2 x SSC containing 0.1% SDS twice. DNA sequence analysis: The plasmids containing the mouse TH cDNAs were treated with exonuclease JII and mung bean nuclease to construct nested deletions of various lengths ( 12 ). Nucleotide sequence was determined by the dideoxynucleotide chain termination method using Sequenase (United States Biochemical Corp., Cleaveland, OH ) ( 13 ). PCR ( polymerase chain reaction ) cloning: Lambda gt 10 forward primer was obtained from Takara Shuzo, Co., LTD ( Kyoto, Japan ). Other oligonucleotides were synthesized by using an Applied biosystems DNA synthesizer. The sequences of primers are shown as follows: S’dGCTGGGTAGTCCCCACC 3’ ( xgt 10 primer ) 5’dTCCAATGGGTTCCCAGGTT 3’( primer A ), S’dGCACTATGCCCACCCCCAGCGC 3’ ( primer B ), 5’dGGACAGCATJTCCATCCCTCTCCTC 3’( primer C ), S’dCCGTGTCTGAGCTGGACGCCAAGCAGGC 3’( primer D ), SdCCTTGCGGGCGTCCTCGATGAGGCTCTG 3’( primer E ). Primers, D and E were designed using human TH sequence. Primers, A, B, and C were either identical or complementary to mouse TH sequence. PCR ( 14 ) was performed in 100 t.t.lof a reaction mixture containing template DNA ( 1 ug of DNA prepared from amplified library or 5 t.tlof fust step PCR reaction mixture ) in 50 mM KCl, 10 mM Tris-HCl ( pH 9.0 ), 1.5 mM MgC12,0.01% gelatin, 0.1% Triton X-100,200 t.tM each of dNTPs ( dATP, dTTP, dCTP, and dGTP ), and a pair of primers ( 20 pmoles each ). The primers and conditions used are shown as follows: pTH-1: first step, hgt 10 forward primer and primer A; second step, hgt 10 forward primer and primer E; 94°C 1 min, 57°C 1 min ,72”C 1 min and 30sec, 30 cycles for both steps. pTH-2: first step, primers B and C, 93-C 2 min, 72°C 1 min, 30 cycles; second step primers C and D, 94” C 1 min, 65” C 1 min and 72°C 1 min and 30sec, 30 cycles. pTH-3: first step, primers B and C, 93” C 2 min, 72°C 1 min, 30 cycles; primers A and B, 94°C 1 min, 57°C 1 mitt, 72°C 1 min and 30 set, 30 cycles. The DNA fragments amplified were separated by agarose gel electrophoresis and cloned into pUCl19 vector directly ( pTH-1 ) or after digestion with Pst I ( pTH-2 and pTH-3 ). RNA blot analysis: Total RNA ( 10 ug ) from mouse adrenal was subjected to electrophotesis on 1.2% agarose gel containing formaldehyde and tmnsblotted onto a nylon membrane ( Hybond-N, Amersham ) ( 11). Hybridization was carried out in 5 x SSPE ( 1 x SSPE contains 0.18 M NaC1,lO mM NaPO4 ( pH 7.7 ) and 1 mM EDTA ) containing 50% formamide, 0.5% SDS, 5 x Denhardt’s solution, 100 ug/ ml of salmon sperm DNA and cDNA fragment labelled with [a-32P]dCTP by a multi-prime labelling method. The blot was washed with 2 x SSC containing 0.1% SDS and with 0.1 x SSC containing 0.1% SDS, each for 15 min at 50°C.
RESULTS cDNA isolation
AND DISCUSSION
and sequencing:
Five positive clones were obtained by screening of lo6 plaques. These were purified, and digested with EcoRI to yield inserts, the largest of which ( LTH- 1 > was 1.7k bp. This insert was subcloned into a Blue Script KS vector ( STRATAGENE, CA ) and its sequence in both orientations was determined. By analogy with rat sequence, it was suggested that hTH-1 was not a full-length clone 1611
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and lacked approximately 150 bp sequence of its 5’ end. Thus, we undertook to derive additional sequence information 5’ to hTH-1 by the PCR cloning. Three experiments were performed to obtain 5’ region of mouse TH cDNA using two steps PCR (MATERIALS
AND METHODS
). For the first step, DNA prepared from the library
was used as a template. Since no obvious bands were observed on agarose gel after 30 cycles of PCR, one-twentieth of reaction mixture was then subjected to second PCR. In the second step, one of two primers used in the fust step was shifted to inner position to improve the specificity of PCR. The other primer was used for the both, first and second steps (Fig. 1 ). In each experiment, only a single species of DNA was amplified after the second step. These cDNAs were cloned in pUCll9 vector ( pTH-1, pTH-2, and pTH-3 ) and their nucleotide sequences were determined. Overlapping sequences of all four clones, including XTH-1, displayed perfect match. Sequence of each cDNA was highly homologus to rat sequence, providing the strong evidence that it is the mouse TH cDNA.
The restriction map and structure of cDNAs obtained are shown in Fig. 1. The
inserts of LTH 1 and pTH 3 were connected at Pst I site and a full-length cDNA was constructed. The cDNA coding for the mouse TH is 1494 bp long with 5 bp 5’ untranslated and 258 bp 3’ untranslated regions. The consensus polyadenylation AATAA precede a stretch of poly (A)+ residues at the 3’ end ( Fig. 2 ).
signal
Only one class of TH m-RNA was observed in RNA blot with an RNA analysis: estimated size of 1.6 kb ( Fig. 3 ). The multiplicity of TH m-RNA was reported in human ( 3,4,5 ) and monkey tissues ( 15 ). These m-RNAs
are constant for the most
part but differ as to the insertion or deletion of 12 and 8 1 bp sequence near the 5’ end. Since the molecular weight differences of these m-RNAs are relatively small, they could not be deteced separately by Northern blot analysis. Thus, we used PCR to detect these
hTH 1
pTH
1
I
t
t-H
-c EA
pTH
2
!,D
-* 6
-
I -
100 bp
pTH 3
Fig. 1. Snuctureandrestrictionmapof mouseTH cDNA. Closed box, protein coding region;arrows,primersusedto obtaincDNAs; H, theregionof cDNA containedin eachclone;l---l, harm.
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1
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1991
Met Pro Thr Pro Ser Ala GC ACT RTG CCC ACC CCC AGC ‘XC
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Ser Ser Pro Gin Pro Lys Gly Phe Arq Arq Ala Val TCC TCG CCA CAG CCC AAG GGC TTC AGA AGA GCC GTC
18 59
19 60
Ser Gl" Gin Asp Thr Lys Gin Ala Gl" Ala "al Thr Ser Pro Arq Phe Ile Gly Arg Arg TCA GAG CAG GAT ACC AAG C&G GCC GAG GCT GTC ACG TCC CCA AGG TTC A T T GGA CGG CGG
38 119
39 120
Gin Ser Le" Ile Glu Asp Ala Arq Lys Gl" Arq Glu Ala Ala Ala Ala Ala Ala Ala Ala C&G AGT CTC ATC GAG GAT GCC CGC A?& GAG CGG GAG GCA GCA GCA GCT GCA GCA GCG GCT
58 179
59 180
Ala Val Ala Ser Ala Gl" Pro Gly &is!? Pro Le" Glu Ala "al GCG GTA GCC TCC GCG GAA CCT GGG AAC CCA T T G GAG GCT Gn;
"al Phe Gl" Gi" Arg Asp GTA TTC GAG GAG AGG GET
78 239
79 240
Gly Asn Ala Val Leu Asn Le" Le" Phe Ser Leu Arg Gly Thr Ly, Pro Ser Ser Le" Ser CGA AAT GCT G T T CTC AX CTG CTC TTC TCC T T G AGG GGT &CA A?,23 CCC TCC TCR CTG TCT
98 299
99 300
Rrq Ala Le" Lys Val Phe Glu Thr Phe Glu Ala Lys Ile His "is Leu CGG GCT T T G AAA GTG T T T GAG KCA T T T GAP. GCC AAT, ATC CAC C.&C TTA
Gl" Thr Arg Pro GAG ACC CGG CCT
119 359
119 360
Ala Gin Arq Pro Leu Ala Gly 5er GCC CAG AGG CC?, CTG GCA GGA WC
Pro "is Le" Glu Tyr Phe Val Ar9 Phe Glu "al Pro CCC CAC CTG GAG TX T T T GTG CGC TTC GAG GTG CCC
138 419
139 420
Ser Gly Rsp Leu Ala Ala Leu Le" Se= Ser Val Arg Arg Val Ser Asp Asp Val Ar9 Ser ACT GGC GAC CT-Z GCT GCC CTC CTC AGT TCT GTG CGT CGG GTG TCT GAC GAT GTG CGC AGT
158 479
159 480
Ala Arq Glu Asp Lys Val Pro Trp Phe Pro Arg Lys "al Ser Gl" Leu Asp Lys Cys His GCC AGA GAG GAC iLAG G T T CCC TGG TTC CCA AGG AAA GTG TC& GAG TTC GAT A&G T G T CAC
178 539
179 540
His Leu Val Thr Lys C.&C CTG GTC ACC MG
19E 599
199 600
Ala Tyr Arq Gin Arq Arq Lys te" Ile Ala GCG T A T CGC C&G CGC CGG AAG CTG A T T GCA
219 660
Pro Ile Pro His Val Gl" Tyr CCA A T T CCC CAC GTG GAA TX
Ala GCC
238 719
239 720
Thr Leu Lys Gly Leu Tyr Ala Thr His Ala Cys Arg Glu His Le" G1" Ala Phe Gin Leu ACG CTG AAG GGC CTC T A T GCT ACC CAT GCC TGC CGG GAA CAC CTG GAG GCT TTC CAG CTT
258 779
259 780
Leu Gl" Arg Tyr Cys Gly Tyr Arq CTG GAI\ CGG TAC T G T GGC TAC cm
278 839
219 840
Phe Leu tys Glu Arg Thr Cly Phe Gin Leu Arg Pro "al Ala Gly Leu Leu 5er Ala Arg TTC T T G ARG G&A CGG ACT GGC TTC CAG CTG CGA CCC GTG GCC GGT CT?, CTG TCT GCC CGT
298 899
299 900
Asp Phe Lc" GAT T T T cm
318 959
319 960
Ser WC
Phe Asp Pro Asp Leu Asp Le" Asp His Pro Gly Phe 5er Asp Gl" T T T GAC CCT GAC CTG GAC CTG GAC CAT CCG GGC TTC TCT GAC CAG Gl" Ile Ala GAG A T T GfC
Phe Gin Tyr Lys Gin Gly Gl" TTC CAR TAC SAG CAG GGT GAG
Thr Lys Glu Gl" Ile Ala Thr Trp Lys Gl" "al Tyr RCA ARC GAG GAA A T T GCT ACC TGG AAG GAG GTA TX
Glu Asp Ser Ile Pro Gin Le" Glu Asp "al Set His GAG GAc RGC A T T cc.4 CAG CTG GAG GAT GTG TCT c*c
Ala Ser Le" Ala Phe Ar9 "al Phe Gl" Cys Thr GCC AGT CTG GCC T T c CGT GTG T T T CAG TGC *ctt
Gin Tyr Ile Arq His Ala CAG TAC ATC CGT C*T GCC
218 659
Ser Pro Met His Ser Pro Gl" Pro Asp Cys Cys His Gl" Le" Le" Gly His Val Pro 338 TCA CCT ATG CRC TCA CCC GAG CCA GAC TGC TGC CAC GAG CTG CTG GGA CAC GTA CCC 1019
339 1020
Met Leu Ala Asp Arq Thr Phe Ala Gl" Phe Ser Gin Asp Ile Gly Leu Ala Ser Le" Gly 358 ATG T T G GCT GAC CGC AC.=. T T T GCC CAG TTC TCC CAG GAC A T T CGA CTT GCA TCT CTG GGG 1079
359 IOEO
Ala Ser Asp Gl" Gl" Ile G1" tys Leu Ser Thr Val Tyr Trp Phe Thr "al Gl" Phe Gly 378 GCT TCA GAT GAA GA.& A T T GAP, PlAA CTC TCC RCG GTG TAC TGG TTC ACT GTG GAG T T T GGG 1139
379 1140
Leu cn;
399 1200
Glu Ix" GAG CTC
419 1260
Ala "al Gin Pro Tyr Gin Asp Gin Thr Tyr Gin Pro "al Tyr Phe Val Ser Gl" SK Phe 438 GCC GTG CAG CCC TAC CAA GAT CAA ACC TAC CAG CCG GTG TAC TTC GTG TCA GAG AGC TTC 1319
439 1320
Ser Asp Ala L,‘s Asp Lys Le" Arg As" Tyr Ala Ser Arg Ile Gin A=9 Pro Phe Ser Val 458 AGT GAT GCC AAG GAC FAG CTC AGG AAC T A T GCC TCT CGT ATC CAG CCC CCA TTC TCT GTG 1379
459 1380
Lys Phe Asp Pro Tyr AAG T T T GAC CCG TX
419 1440
Ser Le" Gl" Gly Val Gin Asp Gl" Le" His Thr Le" Thr Gl" Ala Le" Ser Ala Ile 5er 498 TCC T T A GAG GGG GTC CAG GAT GAG CTG CAC ACC CTG ACC CAA GCA CTG AGT GCC A T T AGC 1499
499 1500
*** TAT, ATG CAT AGG GTA CCA CCC ACA GGT GCC AGG GGC CTT KC
Cys Lys TGT m
Gin cw
As" Gly Cl" IUT GGG em
Leu Lys Ala Tyr Gly Ala Gly Le" Leu Ser Ser CTG w,G GCT T&C GG* GCA GGG CTG CTG TCT xc
Tyr Tm
Gly 398 GGA 1199
Le" His Ser Leu Ser Gl" Gl" Pro Glu Val Afq Ala Phe Asp Pro Asp Thr Ala 418 CTG CAC TCC CTG TCA GAG GAG CCC GAG GTC CGG GCC T T T GAC CCA GAC ACA GCA 1259
Thr Le" Ala Ile Asp "al Leu Asp Ser Pro Hia Thr Ile Arg Arq ACC CTG GCC A T T GAT GTA CTG GAC AGT CCT CAC ACC ATC CGG c‘C
478 1439
499 CAA AGT TCC CAG CCC CTT 1559
1560
CTC CPA CCT TTC CTG GCC C.&G AGG CTT TCC CAT GTG T G T GGC TGG ACC CT-I TGA TGG GCT 1619
1620
GCT CTT GGT CCC CCT CCT CC.4 cTG CTP CT‘? AX
CAC ATC T T A CTA CTG CAT GCG CTC C&G 1679
1680
GAT GGT CCT GCA TTC CTC CTG CCC TTC ATG Cl-G T A T TCT AU.7 CTG A T T A T T ATTC TC-
1739
1740
AAG GAA GGA AAG GTC
1774
TCC
AAA AAA AM,
AA& AApl A?+
Fig. Nucieotide and deduced amino acid sequence of mouse tyrosine hydroxylase. Polyadenylation signal is underlined. 1613
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-18s
11872-
03
04
-Type
2
-Type
1
1234567
Fi .3. Northern blot analysis. Total RNA ( 10 pg ) of mouse adrenal was hybridized
272. P labelled hTH-1 as described in MATERIALS 28s and 18s rRNA are indicated
AND METHODS.
Positions of
Fig. Agarose gel electrophoresis of TH cDNA amplified by PCR. Single stranded cDNA was synthesized using 10 pg of RNA with Murine Molony Leukemia Virus reverse transcriptase ( Bethesda Research Laboratories ) and primer E. Double stranded cDNA were synthesized and amplified using primer D, primer E, and Taq DNA polymerase ( Promega Biotechnology ) for 30 cycles ( 93’C, 2 min and 72” C, lmin ). cDNAs were electrophoresed in 4% Nusieve ( FMC Bioproduct ) GTG agarose gel and stained with ethidium bromide. Lane 1, +X/ HaeIII marker ; lane 2, RNA minus control; lane 3, human type 1 TH cDNA; lane 4 human type 2 TH cDNA; lane 5, Macaca irus, adrenal; lane 6, mouse, adrenal; lane 7, rat, brain.
subtypes. A singlespeciesof m-RNA correspondingto type 1 TH was detectedin RNA from mouseand rat, while two speciesof m-RNA were detectedin monkey tissue ( Fig. 4 ). From theseresults,we suggestthat there are only a singlespeciesof TH mRNA strongly expressedin mouse. Amino acid sequence analysis: The amino acid sequenceof mouseTH is deducedfrom nucleotidesequence.When the first ATG is usedasthe initiation codon, mousePI codesfor 498 amino acidsand hasa calculatedmolecularmassof 55990. Amino acid sequenceof mouseTH is comparedwith thoseof rat and humanenzymes. As shown in Fig 5, sequencesof TH from thesesourceswere highly homologusto each other. The sequenceof mouseTH exhibits 97% and92% homology with rat and human enzymes,respectively. It is known that TH activity is modulatedby severalfactors. Among them, activation of TH by phosphorylationis the mostimportant short term regulation of the enzyme. Major putative phosphorylationsitesof rat TH locatedin the N-terminal region are conservedin mouseTH ( for example, SerSfor prolinedirected protein kinase( 16 ), SerI9 and Ser40 for Ca2+/ calmodulin-dependentprotein kinase, and Se@ and Ser153 for CAMP-dependentprotein kinase) ( 17 ). 1614
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1 1 1
Mouse Rat HUllan Mouse Rat Human
51
51 51
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MPTPSASSPQPKGFRRAVSEQDTKQAEAVTSPRFIGRRQSLIEDARKERE ------P---------------A------------------------------D-TT--A---------L-A-----IM-------------------AAAAAARAAVASAEPGNPLEAVVFEERDGNAVLNLLFSLRGTKPSSLSRA ------------S--------------------------------------V-------P-.---D-----A---KE-N--------p-A----A----
Mouse Rat HUllJaIl
101 101 100
LKVFETFEAKIHHLETRPAQRPLAGSPHLEYFVRFEVPSGDLAALLSSVR v------------------------------------------------V---------------------R--G--------L--RR--------G--
Mouse Rat Human
151
RVSDDVRSAREDKVPWFPRKVSELDKCHHLVTKFDPDLDLDHPGFSDQAY -------------------------------------------------A
Mouse Rat Human
201 201 200
RQRRKLIAEIAFQYKQGEPIPHVEYTKEEIATWKEVYATLKGLYATHACR ---------------H---------A----------"-------------------------RH-D---R---------------T------------
Mouse Rat HUlXltl
251 251 250
EHLEAFQLLERYCGYREDSIPQLEDVSHFLKERTGFQLRPVAGLLSARDF ----G----------------------R-------------------------A-A----FS-----N-------------------------------
Mouse Rat Human
301 301 300
LASLAFRVF,7CTQYIRHASSPMHSPEPDCCHELLGHVPMLADRTFAQFSQ _____________-__--_------------------------------_--___-__-___-__--__------------------------------
Mouse Rat Human
351 351 350
DIGLASLGASDEEIEKLSTVYWFTVEFGLCKQNGELKAYGAGLLSSYGEL _------___-----__---------------------------------------------------LS--------------V--------------
Mouse Rat Human
401 401 400
LHSLSEEPEVRAFDPDTAAVQPYQDQTYQPVYFVSESFSDAKDKLRNYAS --------------------------------------N------------C------I-----EA------------S--------S-------S---
Mouse Rat Human
451 451 450
RIQRPFSVKFDPYTLAIDVLDSPHTIRRSLEGVQDELHTLTQALSAIS
151 150
COMMUNICATIONS
Q--E----pAGp--------------------------------------
--------------------------Q-------------AH------
-----------------------QAVR----------D---------G
Fig. Alignment of amino acid sequences for mouse, rat, and human ( type 1 ) tyrosine hydroxylase. Hyphen, amino acid identical to that of mouse; dot, gap.
ACKNOWLEDGMENTS This work was supported by a Grant -in-Aid for Scientific Research, from the Ministery of Education, Science and Culture, Japan. We thank Dr. Shigeo Ohno for giving us a mouse brain cDNA library. We also thank Dr. Hiroshi Ichinose for helpful discussions.
REFERENCES 1. Nagatsu, T., Levitt, M., and Udenfriend , S. ( 1964 > J. Biol. Chem. m, 2910-2917. 2. Catecholamines: Basic and Peripheral Mechanisms (eds. Usdin, E., Carlsson, A., Dahlstorm, A., and Engel, J. ) (1984 ) Alan R. Liss, Inc., New York. 3. Grima, B., Lamouroux, A., Boni, C., Julien, J.-F., Javoy-Agid, F., and Mallet, J. (1987 ) Nature %,707-711. 4. Kaneda, N., Kobayashi, K., Ichinose, H., Kishi, F., Nakazawa, A., Kurosawa, Y., Fujita, K., and Nagatsu, T. (1987 ) B&hem. Biophys. Res. Commun *-,146 971-975. 5. Kobayashi, K., Kaneda, N., Ichinose, H., Kishi, F., Nakazawa, A., Kurosawa, Y., Fujita, K., and Nagatsu, T. (1987 ) Nucleic Acids Res. 15, 6733. 6. Grima, B., Lamouroux, A., Blanot, F., Biguet, N.F., and Mallet, J. (1985) Proc. Natl. Acad. Sci. USA a.67 I-621. 1615
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7. D’Mello, S. R., Weisberg, E. P., Stachowiak, M. K. , Turzai, L. M., Gioio, A. E., and Kaplan, B. B. (1988 ) J. Neurochem. Res. 19, 440-449. 8. Fauquet, M., Grima, B., Lamourox, A., and Mallet J. ( 1988 ) J. Neurochem. 3, 142-148. 9. Neckameyer, W. S. , and Quinn, W. G. (1989 ) Neuron 2, 1167- 1175. 10. Kaneda,-N., Sasaoka, T., Xobayashi, K., K&hi, K., Nagatsu, I., Kurosawa, Y., Fujita, K., Yokoyama, M., Nomura, T., Katsuiki, M., and Nagatsu, T. ( 1991 ) Neuron, 6, 1-12. 11. Maniatis, T., Fritsch, E. F., and Sambrook, J. ( 1989 ) Molecular Cloning: A Laboratory Mammal, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 12. Heinkoff, S. ( 1984 ) Gene &351-359. 13. Sanger, F., Nicklen, S., and Coulson, A. R. ( 1977 ), Proc. Natl. Acad Sci. USA B,54635467. 14. Saiki, R. K. Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A. and Amheim, N. ( 1985 ) Science 230, 1350-1354. 15. Ichikawa, S., Ichinose, H., and Nagatsu, T., ( 1990 ) B&hem. Biophys. Res. Commun. 173, 1331-1336. 16. Vulliet, P. R., Hall, F. L., Mitchell, J. P., and Hardie, D. G. ( 1989 ) J. Biol. Chem. a, 16292-16298. 17. Zigmond, R. E., Schwartschild, M. A., and Ritten house., A. R. ( 1989 ) Ann. Rev. Neurosci. 12.415-46 1.
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PRIMARY STRUCTURE OF MOUSE TYROSINE HYDROXYLASE DEDUCED FROM ITS cDNA Shinichi Ichikawa, Toshikuni Sasaoka,and ToshiharuNagatsu* Departmentof Biochemistry, Nagoya University School of Medicine, Nagoya 466, Japan Received
April
12,
1991
SUMMARY: The cDNAs for tyrosine hydroxylase were cloned from a mousebrain cDNA library by plaquehybridization. Sincethe IongestcDNA clone lacked approximately 150 bp sequenceof its N-terminal region, additional 5’ region was obtainedusingpolymerasechainreaction. Nucleotidesequencedeterminationof cDNAs revealedthat mousetyrosine hydroxylase m-RNA encodes498 amino acidswith a calculatedmolecularmassof 55990. The aminoacid sequenceof mousetyrosine hydroxylase is highly homologusto rat ( 97% ) andhuman( 92% ) enzymes. o 1991 Academic
Press,
Inc.
Tyrosine hydroxylase ( tyrosine 3-monooxygenase;tyrosine, tetrahydropteridine: oxygen oxidoreductase( 3-hydroxylating ), EC l-14.16.2.) ( TH ) catalyzes the fist and rate-limiting stepin catecholaminebiosynthesis( 1 ). This enzyme is expressedin catecholaminergicneuronsin the discreteregionsof the brain, noradrenergicneuronsof sympatheticgangliaand sympatheticnerves,and noradrenalineand adrenalinecells of adrenalmedulla. TH plays a central role in neuronaltransmissionand hormonalaction of catecholamines( 2 ). The cDNA cloning of TH for human ( 3,4,5 ), rat ( 6 ), bovine ( 7 ), quail ( 8 ), and fruit fly ( 9 ) have already beenreported. On the other hand,a laboratory mouseis one of the mostimportant animalsasa standardsystem, and the study of the mousegeneticsbecomeincreasinglyimportant by the advent of transgenicbiology. Recently, transgenicmice over-expressinghumanTH hasbeenproduced ( 10 ). However, mouseTH hasnot beenstudiedintensively. In this communication,we report on the cDNA cloning of mouseTH and the determinationof its nucleotidesequence.
MATERIALS
AND METHODS
Library screening: A mousebrain cDNA library ( strain ICR, 7 weeksold ) in hgt 10 vector was a generousgift from Dr. ShigeoOhno, Tokyo Metropolitan Institute of Medical Science. A humanTH cDNA ( type 4 ) waslabeledwith [a-P32ldCTP ( 3000 Ci / m mol ) by Multiprime DNA labelling system( Amex-sham) and usedas a probe ( 11 * To whom all correspondenceshouldbe addressed. 0006-291X/91 Copyright All rights
$1.50
0 1991 by Academic Press, of reproduction in any form
OK. reserved.
1610
Vol.
176,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
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). A total of 106 k phage plaques on E.&i strain C6CO h~7 lawn cells were screened. The plaques were Lifted onto nylon membranes ( Hybond-N, Amersham ). The membranes were prehybridized for lhr at 42°C in a buffer containing 6 x SSC ( 1 x SSC is 0.15 M NaCl and 0.015 M sodium citrate ), 0.1% SDS, 5 x Denhartdt’s solution, 50% formamide, and salmon sperm DNA (100 pg./ ml ). Hybridization followed at 42°C for 24 hrs in the above specified buffer with the labeled human TH cDNA ( type 4 ). The membranes were washed at 42” C with 2 x SSC containing 0.1% SDS twice. DNA sequence analysis: The plasmids containing the mouse TH cDNAs were treated with exonuclease JII and mung bean nuclease to construct nested deletions of various lengths ( 12 ). Nucleotide sequence was determined by the dideoxynucleotide chain termination method using Sequenase (United States Biochemical Corp., Cleaveland, OH ) ( 13 ). PCR ( polymerase chain reaction ) cloning: Lambda gt 10 forward primer was obtained from Takara Shuzo, Co., LTD ( Kyoto, Japan ). Other oligonucleotides were synthesized by using an Applied biosystems DNA synthesizer. The sequences of primers are shown as follows: S’dGCTGGGTAGTCCCCACC 3’ ( xgt 10 primer ) 5’dTCCAATGGGTTCCCAGGTT 3’( primer A ), S’dGCACTATGCCCACCCCCAGCGC 3’ ( primer B ), 5’dGGACAGCATJTCCATCCCTCTCCTC 3’( primer C ), S’dCCGTGTCTGAGCTGGACGCCAAGCAGGC 3’( primer D ), SdCCTTGCGGGCGTCCTCGATGAGGCTCTG 3’( primer E ). Primers, D and E were designed using human TH sequence. Primers, A, B, and C were either identical or complementary to mouse TH sequence. PCR ( 14 ) was performed in 100 t.t.lof a reaction mixture containing template DNA ( 1 ug of DNA prepared from amplified library or 5 t.tlof fust step PCR reaction mixture ) in 50 mM KCl, 10 mM Tris-HCl ( pH 9.0 ), 1.5 mM MgC12,0.01% gelatin, 0.1% Triton X-100,200 t.tM each of dNTPs ( dATP, dTTP, dCTP, and dGTP ), and a pair of primers ( 20 pmoles each ). The primers and conditions used are shown as follows: pTH-1: first step, hgt 10 forward primer and primer A; second step, hgt 10 forward primer and primer E; 94°C 1 min, 57°C 1 min ,72”C 1 min and 30sec, 30 cycles for both steps. pTH-2: first step, primers B and C, 93-C 2 min, 72°C 1 min, 30 cycles; second step primers C and D, 94” C 1 min, 65” C 1 min and 72°C 1 min and 30sec, 30 cycles. pTH-3: first step, primers B and C, 93” C 2 min, 72°C 1 min, 30 cycles; primers A and B, 94°C 1 min, 57°C 1 mitt, 72°C 1 min and 30 set, 30 cycles. The DNA fragments amplified were separated by agarose gel electrophoresis and cloned into pUCl19 vector directly ( pTH-1 ) or after digestion with Pst I ( pTH-2 and pTH-3 ). RNA blot analysis: Total RNA ( 10 ug ) from mouse adrenal was subjected to electrophotesis on 1.2% agarose gel containing formaldehyde and tmnsblotted onto a nylon membrane ( Hybond-N, Amersham ) ( 11). Hybridization was carried out in 5 x SSPE ( 1 x SSPE contains 0.18 M NaC1,lO mM NaPO4 ( pH 7.7 ) and 1 mM EDTA ) containing 50% formamide, 0.5% SDS, 5 x Denhardt’s solution, 100 ug/ ml of salmon sperm DNA and cDNA fragment labelled with [a-32P]dCTP by a multi-prime labelling method. The blot was washed with 2 x SSC containing 0.1% SDS and with 0.1 x SSC containing 0.1% SDS, each for 15 min at 50°C.
RESULTS cDNA isolation
AND DISCUSSION
and sequencing:
Five positive clones were obtained by screening of lo6 plaques. These were purified, and digested with EcoRI to yield inserts, the largest of which ( LTH- 1 > was 1.7k bp. This insert was subcloned into a Blue Script KS vector ( STRATAGENE, CA ) and its sequence in both orientations was determined. By analogy with rat sequence, it was suggested that hTH-1 was not a full-length clone 1611
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and lacked approximately 150 bp sequence of its 5’ end. Thus, we undertook to derive additional sequence information 5’ to hTH-1 by the PCR cloning. Three experiments were performed to obtain 5’ region of mouse TH cDNA using two steps PCR (MATERIALS
AND METHODS
). For the first step, DNA prepared from the library
was used as a template. Since no obvious bands were observed on agarose gel after 30 cycles of PCR, one-twentieth of reaction mixture was then subjected to second PCR. In the second step, one of two primers used in the fust step was shifted to inner position to improve the specificity of PCR. The other primer was used for the both, first and second steps (Fig. 1 ). In each experiment, only a single species of DNA was amplified after the second step. These cDNAs were cloned in pUCll9 vector ( pTH-1, pTH-2, and pTH-3 ) and their nucleotide sequences were determined. Overlapping sequences of all four clones, including XTH-1, displayed perfect match. Sequence of each cDNA was highly homologus to rat sequence, providing the strong evidence that it is the mouse TH cDNA.
The restriction map and structure of cDNAs obtained are shown in Fig. 1. The
inserts of LTH 1 and pTH 3 were connected at Pst I site and a full-length cDNA was constructed. The cDNA coding for the mouse TH is 1494 bp long with 5 bp 5’ untranslated and 258 bp 3’ untranslated regions. The consensus polyadenylation AATAA precede a stretch of poly (A)+ residues at the 3’ end ( Fig. 2 ).
signal
Only one class of TH m-RNA was observed in RNA blot with an RNA analysis: estimated size of 1.6 kb ( Fig. 3 ). The multiplicity of TH m-RNA was reported in human ( 3,4,5 ) and monkey tissues ( 15 ). These m-RNAs
are constant for the most
part but differ as to the insertion or deletion of 12 and 8 1 bp sequence near the 5’ end. Since the molecular weight differences of these m-RNAs are relatively small, they could not be deteced separately by Northern blot analysis. Thus, we used PCR to detect these
hTH 1
pTH
1
I
t
t-H
-c EA
pTH
2
!,D
-* 6
-
I -
100 bp
pTH 3
Fig. 1. Snuctureandrestrictionmapof mouseTH cDNA. Closed box, protein coding region;arrows,primersusedto obtaincDNAs; H, theregionof cDNA containedin eachclone;l---l, harm.
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Met Pro Thr Pro Ser Ala GC ACT RTG CCC ACC CCC AGC ‘XC
AND
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Ser Ser Pro Gin Pro Lys Gly Phe Arq Arq Ala Val TCC TCG CCA CAG CCC AAG GGC TTC AGA AGA GCC GTC
18 59
19 60
Ser Gl" Gin Asp Thr Lys Gin Ala Gl" Ala "al Thr Ser Pro Arq Phe Ile Gly Arg Arg TCA GAG CAG GAT ACC AAG C&G GCC GAG GCT GTC ACG TCC CCA AGG TTC A T T GGA CGG CGG
38 119
39 120
Gin Ser Le" Ile Glu Asp Ala Arq Lys Gl" Arq Glu Ala Ala Ala Ala Ala Ala Ala Ala C&G AGT CTC ATC GAG GAT GCC CGC A?& GAG CGG GAG GCA GCA GCA GCT GCA GCA GCG GCT
58 179
59 180
Ala Val Ala Ser Ala Gl" Pro Gly &is!? Pro Le" Glu Ala "al GCG GTA GCC TCC GCG GAA CCT GGG AAC CCA T T G GAG GCT Gn;
"al Phe Gl" Gi" Arg Asp GTA TTC GAG GAG AGG GET
78 239
79 240
Gly Asn Ala Val Leu Asn Le" Le" Phe Ser Leu Arg Gly Thr Ly, Pro Ser Ser Le" Ser CGA AAT GCT G T T CTC AX CTG CTC TTC TCC T T G AGG GGT &CA A?,23 CCC TCC TCR CTG TCT
98 299
99 300
Rrq Ala Le" Lys Val Phe Glu Thr Phe Glu Ala Lys Ile His "is Leu CGG GCT T T G AAA GTG T T T GAG KCA T T T GAP. GCC AAT, ATC CAC C.&C TTA
Gl" Thr Arg Pro GAG ACC CGG CCT
119 359
119 360
Ala Gin Arq Pro Leu Ala Gly 5er GCC CAG AGG CC?, CTG GCA GGA WC
Pro "is Le" Glu Tyr Phe Val Ar9 Phe Glu "al Pro CCC CAC CTG GAG TX T T T GTG CGC TTC GAG GTG CCC
138 419
139 420
Ser Gly Rsp Leu Ala Ala Leu Le" Se= Ser Val Arg Arg Val Ser Asp Asp Val Ar9 Ser ACT GGC GAC CT-Z GCT GCC CTC CTC AGT TCT GTG CGT CGG GTG TCT GAC GAT GTG CGC AGT
158 479
159 480
Ala Arq Glu Asp Lys Val Pro Trp Phe Pro Arg Lys "al Ser Gl" Leu Asp Lys Cys His GCC AGA GAG GAC iLAG G T T CCC TGG TTC CCA AGG AAA GTG TC& GAG TTC GAT A&G T G T CAC
178 539
179 540
His Leu Val Thr Lys C.&C CTG GTC ACC MG
19E 599
199 600
Ala Tyr Arq Gin Arq Arq Lys te" Ile Ala GCG T A T CGC C&G CGC CGG AAG CTG A T T GCA
219 660
Pro Ile Pro His Val Gl" Tyr CCA A T T CCC CAC GTG GAA TX
Ala GCC
238 719
239 720
Thr Leu Lys Gly Leu Tyr Ala Thr His Ala Cys Arg Glu His Le" G1" Ala Phe Gin Leu ACG CTG AAG GGC CTC T A T GCT ACC CAT GCC TGC CGG GAA CAC CTG GAG GCT TTC CAG CTT
258 779
259 780
Leu Gl" Arg Tyr Cys Gly Tyr Arq CTG GAI\ CGG TAC T G T GGC TAC cm
278 839
219 840
Phe Leu tys Glu Arg Thr Cly Phe Gin Leu Arg Pro "al Ala Gly Leu Leu 5er Ala Arg TTC T T G ARG G&A CGG ACT GGC TTC CAG CTG CGA CCC GTG GCC GGT CT?, CTG TCT GCC CGT
298 899
299 900
Asp Phe Lc" GAT T T T cm
318 959
319 960
Ser WC
Phe Asp Pro Asp Leu Asp Le" Asp His Pro Gly Phe 5er Asp Gl" T T T GAC CCT GAC CTG GAC CTG GAC CAT CCG GGC TTC TCT GAC CAG Gl" Ile Ala GAG A T T GfC
Phe Gin Tyr Lys Gin Gly Gl" TTC CAR TAC SAG CAG GGT GAG
Thr Lys Glu Gl" Ile Ala Thr Trp Lys Gl" "al Tyr RCA ARC GAG GAA A T T GCT ACC TGG AAG GAG GTA TX
Glu Asp Ser Ile Pro Gin Le" Glu Asp "al Set His GAG GAc RGC A T T cc.4 CAG CTG GAG GAT GTG TCT c*c
Ala Ser Le" Ala Phe Ar9 "al Phe Gl" Cys Thr GCC AGT CTG GCC T T c CGT GTG T T T CAG TGC *ctt
Gin Tyr Ile Arq His Ala CAG TAC ATC CGT C*T GCC
218 659
Ser Pro Met His Ser Pro Gl" Pro Asp Cys Cys His Gl" Le" Le" Gly His Val Pro 338 TCA CCT ATG CRC TCA CCC GAG CCA GAC TGC TGC CAC GAG CTG CTG GGA CAC GTA CCC 1019
339 1020
Met Leu Ala Asp Arq Thr Phe Ala Gl" Phe Ser Gin Asp Ile Gly Leu Ala Ser Le" Gly 358 ATG T T G GCT GAC CGC AC.=. T T T GCC CAG TTC TCC CAG GAC A T T CGA CTT GCA TCT CTG GGG 1079
359 IOEO
Ala Ser Asp Gl" Gl" Ile G1" tys Leu Ser Thr Val Tyr Trp Phe Thr "al Gl" Phe Gly 378 GCT TCA GAT GAA GA.& A T T GAP, PlAA CTC TCC RCG GTG TAC TGG TTC ACT GTG GAG T T T GGG 1139
379 1140
Leu cn;
399 1200
Glu Ix" GAG CTC
419 1260
Ala "al Gin Pro Tyr Gin Asp Gin Thr Tyr Gin Pro "al Tyr Phe Val Ser Gl" SK Phe 438 GCC GTG CAG CCC TAC CAA GAT CAA ACC TAC CAG CCG GTG TAC TTC GTG TCA GAG AGC TTC 1319
439 1320
Ser Asp Ala L,‘s Asp Lys Le" Arg As" Tyr Ala Ser Arg Ile Gin A=9 Pro Phe Ser Val 458 AGT GAT GCC AAG GAC FAG CTC AGG AAC T A T GCC TCT CGT ATC CAG CCC CCA TTC TCT GTG 1379
459 1380
Lys Phe Asp Pro Tyr AAG T T T GAC CCG TX
419 1440
Ser Le" Gl" Gly Val Gin Asp Gl" Le" His Thr Le" Thr Gl" Ala Le" Ser Ala Ile 5er 498 TCC T T A GAG GGG GTC CAG GAT GAG CTG CAC ACC CTG ACC CAA GCA CTG AGT GCC A T T AGC 1499
499 1500
*** TAT, ATG CAT AGG GTA CCA CCC ACA GGT GCC AGG GGC CTT KC
Cys Lys TGT m
Gin cw
As" Gly Cl" IUT GGG em
Leu Lys Ala Tyr Gly Ala Gly Le" Leu Ser Ser CTG w,G GCT T&C GG* GCA GGG CTG CTG TCT xc
Tyr Tm
Gly 398 GGA 1199
Le" His Ser Leu Ser Gl" Gl" Pro Glu Val Afq Ala Phe Asp Pro Asp Thr Ala 418 CTG CAC TCC CTG TCA GAG GAG CCC GAG GTC CGG GCC T T T GAC CCA GAC ACA GCA 1259
Thr Le" Ala Ile Asp "al Leu Asp Ser Pro Hia Thr Ile Arg Arq ACC CTG GCC A T T GAT GTA CTG GAC AGT CCT CAC ACC ATC CGG c‘C
478 1439
499 CAA AGT TCC CAG CCC CTT 1559
1560
CTC CPA CCT TTC CTG GCC C.&G AGG CTT TCC CAT GTG T G T GGC TGG ACC CT-I TGA TGG GCT 1619
1620
GCT CTT GGT CCC CCT CCT CC.4 cTG CTP CT‘? AX
CAC ATC T T A CTA CTG CAT GCG CTC C&G 1679
1680
GAT GGT CCT GCA TTC CTC CTG CCC TTC ATG Cl-G T A T TCT AU.7 CTG A T T A T T ATTC TC-
1739
1740
AAG GAA GGA AAG GTC
1774
TCC
AAA AAA AM,
AA& AApl A?+
Fig. Nucieotide and deduced amino acid sequence of mouse tyrosine hydroxylase. Polyadenylation signal is underlined. 1613
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-18s
11872-
03
04
-Type
2
-Type
1
1234567
Fi .3. Northern blot analysis. Total RNA ( 10 pg ) of mouse adrenal was hybridized
272. P labelled hTH-1 as described in MATERIALS 28s and 18s rRNA are indicated
AND METHODS.
Positions of
Fig. Agarose gel electrophoresis of TH cDNA amplified by PCR. Single stranded cDNA was synthesized using 10 pg of RNA with Murine Molony Leukemia Virus reverse transcriptase ( Bethesda Research Laboratories ) and primer E. Double stranded cDNA were synthesized and amplified using primer D, primer E, and Taq DNA polymerase ( Promega Biotechnology ) for 30 cycles ( 93’C, 2 min and 72” C, lmin ). cDNAs were electrophoresed in 4% Nusieve ( FMC Bioproduct ) GTG agarose gel and stained with ethidium bromide. Lane 1, +X/ HaeIII marker ; lane 2, RNA minus control; lane 3, human type 1 TH cDNA; lane 4 human type 2 TH cDNA; lane 5, Macaca irus, adrenal; lane 6, mouse, adrenal; lane 7, rat, brain.
subtypes. A singlespeciesof m-RNA correspondingto type 1 TH was detectedin RNA from mouseand rat, while two speciesof m-RNA were detectedin monkey tissue ( Fig. 4 ). From theseresults,we suggestthat there are only a singlespeciesof TH mRNA strongly expressedin mouse. Amino acid sequence analysis: The amino acid sequenceof mouseTH is deducedfrom nucleotidesequence.When the first ATG is usedasthe initiation codon, mousePI codesfor 498 amino acidsand hasa calculatedmolecularmassof 55990. Amino acid sequenceof mouseTH is comparedwith thoseof rat and humanenzymes. As shown in Fig 5, sequencesof TH from thesesourceswere highly homologusto each other. The sequenceof mouseTH exhibits 97% and92% homology with rat and human enzymes,respectively. It is known that TH activity is modulatedby severalfactors. Among them, activation of TH by phosphorylationis the mostimportant short term regulation of the enzyme. Major putative phosphorylationsitesof rat TH locatedin the N-terminal region are conservedin mouseTH ( for example, SerSfor prolinedirected protein kinase( 16 ), SerI9 and Ser40 for Ca2+/ calmodulin-dependentprotein kinase, and Se@ and Ser153 for CAMP-dependentprotein kinase) ( 17 ). 1614
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1 1 1
Mouse Rat HUllan Mouse Rat Human
51
51 51
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MPTPSASSPQPKGFRRAVSEQDTKQAEAVTSPRFIGRRQSLIEDARKERE ------P---------------A------------------------------D-TT--A---------L-A-----IM-------------------AAAAAARAAVASAEPGNPLEAVVFEERDGNAVLNLLFSLRGTKPSSLSRA ------------S--------------------------------------V-------P-.---D-----A---KE-N--------p-A----A----
Mouse Rat HUllJaIl
101 101 100
LKVFETFEAKIHHLETRPAQRPLAGSPHLEYFVRFEVPSGDLAALLSSVR v------------------------------------------------V---------------------R--G--------L--RR--------G--
Mouse Rat Human
151
RVSDDVRSAREDKVPWFPRKVSELDKCHHLVTKFDPDLDLDHPGFSDQAY -------------------------------------------------A
Mouse Rat Human
201 201 200
RQRRKLIAEIAFQYKQGEPIPHVEYTKEEIATWKEVYATLKGLYATHACR ---------------H---------A----------"-------------------------RH-D---R---------------T------------
Mouse Rat HUlXltl
251 251 250
EHLEAFQLLERYCGYREDSIPQLEDVSHFLKERTGFQLRPVAGLLSARDF ----G----------------------R-------------------------A-A----FS-----N-------------------------------
Mouse Rat Human
301 301 300
LASLAFRVF,7CTQYIRHASSPMHSPEPDCCHELLGHVPMLADRTFAQFSQ _____________-__--_------------------------------_--___-__-___-__--__------------------------------
Mouse Rat Human
351 351 350
DIGLASLGASDEEIEKLSTVYWFTVEFGLCKQNGELKAYGAGLLSSYGEL _------___-----__---------------------------------------------------LS--------------V--------------
Mouse Rat Human
401 401 400
LHSLSEEPEVRAFDPDTAAVQPYQDQTYQPVYFVSESFSDAKDKLRNYAS --------------------------------------N------------C------I-----EA------------S--------S-------S---
Mouse Rat Human
451 451 450
RIQRPFSVKFDPYTLAIDVLDSPHTIRRSLEGVQDELHTLTQALSAIS
151 150
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Q--E----pAGp--------------------------------------
--------------------------Q-------------AH------
-----------------------QAVR----------D---------G
Fig. Alignment of amino acid sequences for mouse, rat, and human ( type 1 ) tyrosine hydroxylase. Hyphen, amino acid identical to that of mouse; dot, gap.
ACKNOWLEDGMENTS This work was supported by a Grant -in-Aid for Scientific Research, from the Ministery of Education, Science and Culture, Japan. We thank Dr. Shigeo Ohno for giving us a mouse brain cDNA library. We also thank Dr. Hiroshi Ichinose for helpful discussions.
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7. D’Mello, S. R., Weisberg, E. P., Stachowiak, M. K. , Turzai, L. M., Gioio, A. E., and Kaplan, B. B. (1988 ) J. Neurochem. Res. 19, 440-449. 8. Fauquet, M., Grima, B., Lamourox, A., and Mallet J. ( 1988 ) J. Neurochem. 3, 142-148. 9. Neckameyer, W. S. , and Quinn, W. G. (1989 ) Neuron 2, 1167- 1175. 10. Kaneda,-N., Sasaoka, T., Xobayashi, K., K&hi, K., Nagatsu, I., Kurosawa, Y., Fujita, K., Yokoyama, M., Nomura, T., Katsuiki, M., and Nagatsu, T. ( 1991 ) Neuron, 6, 1-12. 11. Maniatis, T., Fritsch, E. F., and Sambrook, J. ( 1989 ) Molecular Cloning: A Laboratory Mammal, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 12. Heinkoff, S. ( 1984 ) Gene &351-359. 13. Sanger, F., Nicklen, S., and Coulson, A. R. ( 1977 ), Proc. Natl. Acad Sci. USA B,54635467. 14. Saiki, R. K. Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A. and Amheim, N. ( 1985 ) Science 230, 1350-1354. 15. Ichikawa, S., Ichinose, H., and Nagatsu, T., ( 1990 ) B&hem. Biophys. Res. Commun. 173, 1331-1336. 16. Vulliet, P. R., Hall, F. L., Mitchell, J. P., and Hardie, D. G. ( 1989 ) J. Biol. Chem. a, 16292-16298. 17. Zigmond, R. E., Schwartschild, M. A., and Ritten house., A. R. ( 1989 ) Ann. Rev. Neurosci. 12.415-46 1.
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