Genes for catecholamine biosynthesis : cloning by expression and identification of the cDNA for rat dopamine beta-hydroxylase
O'MALLEY, K. L., et al.
mRNA for dopamine beta-hydroxylase [3,4-dihydroxyphenylethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 18.104.22.168] has been partially purified from poly(A)+
mRNA isolated from a rat pheochromocytoma cell line. Shared antigenic determinants between tyrosine hydroxylase and dopamine beta-hydroxylase allowed us to obtain enriched fractions of dopamine beta-hydroxylase mRNA by immunoprecipitating translated mRNA products with tyrosine hydroxylase antisera. The enriched dopamine beta-hydroxylase mRNA was used to synthesize the corresponding cDNAs, which were then cloned in the Pst I site of pBR322. Recombinant colonies were characterized by an in situ colony immunoassay and hybrid-selected translation. In vitro translation of the mRNA selected from one recombinant clone produced a protein of 75,000 daltons that comigrated with authentic dopamine beta-hydroxylase. Partial proteolysis of both authentic dopamine beta-hydroxylase and the protein encoded by the recombinant clone produced identical peptide patterns.
O'MALLEY, K. L., et al. Genes for catecholamine biosynthesis : cloning by expression and identification of the cDNA for rat dopamine beta-hydroxylase. Proceedings of the National Academy of Sciences, 1983, vol. 80, no. 8, p. 2161-2165
PMID : 6572968
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Genes for catecholamine biosynthesis: Cloning by expression and identification of the cDNA for rat dopamine e,-hydroxylase
(recombinant DNA/antigenic determinants/insituimmunoassay/hybrid-selected translation)
BARCHASt,AND LARRY KEDES*t§
*theNancyPritzkerLaboratoryofBehavioral Neurochemistry, Department of Psychiatry and Behavioral Sciences,StanfordUniversitySchoolof Medicine,Stanlbrd,California 94305; and tVeteransAdministrationMedicalCenter, PaloAlto,California 94304
Communicated by RichardF.Thompson, January 10, 1983
ABSTRACT mRNA for dopamine 3-hydroxylase [3,4-dihy- droxyphenylethylamine, ascorbate:oxygen oxidoreductase (fl-hy- droxylating), EC 22.214.171.124] has been partially purified from poly(A)+ mRNA isolated from a ratpheochromocytoma cell line.
Sharedantigenic determinants between tyrosine hydroxylase and dopamine f-hydroxylase allowed us to obtain enriched fractions ofdopamine13hydroxylasemRNAbyimmunoprecipitating trans- lated mRNAproducts with tyrosinehydroxylase antisera. The en- richeddopamine(-hydroxylasemRNA wasused to synthesize the corresponding cDNAs, which were then cloned in the Pst I site of pBR322. Recombinant colonies were characterized by an in situ colony immunoassay and hybrid-selected translation. In vitro translation ofthe mRNA selected from one recombinant clone produced a protein of 75,000 daltons thatcomigrated with au- thenticdopamine 3-hydroxylase. Partial proteolysis of both au- thenticdopamine 3-hydroxylaseand theprotein encoded by the recombinant cloneproduced identical peptide patterns.
Thecatecholamines dopamine, epinephrine, and norepineph- rine areformed from theiraminoacidprecursor tyrosine in brain, chromaffincells,and sympatheticnervesandganglia. Enzymes involved in this pathway include tyrosine hydroxylase (Tyr- OHase), DOPAdecarboxylase,
3,4-dihydroxyphenylethylamine,ascorbate:oxygen oxidoreduc- tase
(3-hydroxylating),EC126.96.36.199], and phenylethanolamine methyltransferase (PhEtnMeTase). Althoughlittle isknownabout the cellularand molecularmechanisms governingthelong-term regulation of theseenzymes,
glucocorticoids(1-3), neuralstim- ulation (4), andnerve growth factor (2, 5-6)canall influence the expressionofTyrOHase. Such studies have beenfacilitated bythedevelopment ofaratpheochromocytoma cellline PC 12, whichproduces relatively largeamountsof the catecholamine pathway enzymes(5, 7).
Johetal. (8) suggested considerable homologybetweenTyrOHase, DBH,andPhEtnMeTasebased on
peptideanalysis andantisera crossreactivity. In
particular,Johetal.demonstrated that antibodies raisedagainstTyrOHase precipitatedDBHandPhEtnMeTaseaswell.Peptidemapping bypartialproteolysis and gel electrophoresis indicatedthat DBH, TyrOHase, and PhEtnMeTase had identical
peptidesincom- mon, butnoprimaryaminoacid sequence data has yet been reported.
Shared antigenic determinants andpeptidepatternsnotonly suggestsimilarprimary structure amongthese proteins but also structural homologiesat the nucleotide level. Such interrela- tionships mightbe exploitedtoisolate
specificmRNAsfor the catecholaminepathwayenzymes. For
example,available anti- serafor one enzymecanbe usedtopurifynotjustitsown mRNA
but also a crossreactingenzyme'smRNA.This can beachieved by translatingfractionatedmRNAs inan invitroprotein-trans- lating systemand identifying theproducts by immunoprecip- itationwiththecrossreacting antisera. The mRNA fraction en- riched in the species of interest can then be used to prepare complementary DNAclones encoding the crossreacting pro- tein.
Wehaveused such a scheme to isolate mRNA from the PC 12clonal celllineand haveusedTyrOHase antiserafor theclon- ing and screening procedure by whicha DBH clone was se- lected. We madepositive identificationofthe cDNA cloneby itsabilitytoselectivelyhybridizewith DBH mRNA.
Materials. Avianmyeloblastosisvirusreversetranscriptase, terminal
deoxyribonucleotidyltransferase,and Pst I werepur- chased from P-L Biochemicals. Oligo(dT)-cellulose and oligo-
(dG)12-18wereobtained from Collaborative Research
(Wal-tham, MA). Staphylococcus aureus protein AandCNBr-acti- vated Sepharose4Bwereobtained fromPharmacia. BA-85 ni- trocellulose filterswereobtained from Schleicher and Schuell.
Restriction endonucleaseswerepurchased from NewEngland
lysate,Bolton-Hunter reagent (2,000Ci/mmol; 1 Ci = 3.7 x
EN3HANCEwasobtained from New England Nuclear.
Purified DBH was
providedby S. Watson, University of Chicago. TyrOHase was purified fromratpheochromocytoma (9),andspecific antibodieswereraisedagainstthe enzyme. An- tibody
specificityagainst TyrOHase was established by im- munoelectrophoresis, and localization ofTyrOHase wasestab- lished by immunohistochemistry in catecholamine-containing neuronsinratbrain
pheochromo-cytomacell linePC 12was
Shooter,Stanford University. Cell linesweremaintainedinDulbecco's modified
Eagle'stissue culture medium
supplementedwith 5% horse serumand 10% fetalcalfserum. Cultureswere maintainedin 12%
C02/88% 02at 370C. Cultures were stimulated by the addition ofdexamethasonetothemediumat 10
,uMfinal con- centration. Freshmedium and dexamethasonewereadded
Isolation ofPoly(A)+mRNAand Enrichment forDBHMes- sages. ExtractionofRNAfrom the PC 12cell linewas
bythe procedureof Strohmanetal.(10). Total
gradient.Frac- Abbreviations: TyrOHase, tyrosine hydroxylase; DBH, Dopamine(3 hydroxylase; PhEtnMeTase,phenylethanolamine methyltransferase.
§Towhom correspondenceshouldbe addressedat:The MEDIGEN Project, 151M, VeteransAdministration MedicalCenter, PaloAlto,
2161 Thepublicationcostsofthisarticleweredefrayedinpartbypagecharge payment.Thisarticlemustthereforebeherebymarked"advertisement"
inaccordancewith18 U. S. C. §1734solelytoindicatethisfact.
2162 Biochemistry: O'Malley et al.
tions wereassayed for DBH mRNA by in vitro translation with a rabbit reticulocyte lysate system (11). Labeled translation products were immunoprecipitated (12), electrophoresed on NaDodSO4 gels(13), and fluorographed (14).
Construction of cDNA Clones. Sucrose gradient fractions containing maximum DBH mRNA activity were pooled, and themRNAwasused for thesynthesis of double-stranded cDNA by the procedure of Land et al.(15). After a dCTP-tailing step, the double-stranded cDNA was hybridized to pBR322 tailed withdGTP at the Pst I site. The inserted plasmid wasusedto transform Escherichia coli C600rk mk . Ampicillin-sensitive, tetracycline-resistant clones were transferred tonitrocellulose filters on agar plates. All recombinant DNA procedures fol- lowedthe pertinent NationalInstitutes of Healthguidelines.
In Situ Colony Immunoassay. Colonies grown on nitrocel- lulosefilters werepreparedforcolonyimmunoassayessentially asdescribedbyKemp andCowman(16). Bacterial colonies were lysedin situ,and proteins were bound to activated CNBr paper (17). Covalentlybound antigens were detected by incubating withantiserafollowedby '25I-labeledproteinAfromS.aureus.
In order to remove antibacterial antibodies, rabbitTyrOHase antiserum was passedover acolumnofE. coliC600/pBR322 protein extractboundto aCNBr-Sepharose4Bcolumn(18). S.
aureus protein A wasiodinatedwith'25I-labeled Bolton-Hunter reagent(19) to aspecificactivity of1 X
Positive mRNASelection. Plasmid DNA wasisolated from immunoreactiveclones, CsCl-purified, andboundtonitrocel- lulose filters by modifications of the method of Ricciardietal.
(20). Plasmid DNA(10 ,ug)wasdenaturedin 2 M NaCl/0.2M NaOH,briefly heatedat90°C,chilled,andimmediatelyloaded ontonitrocellulose filters. Prior to
loadingthe DNA, filterswere
1 2 3 4
FIG. 1. Immunoprecipitationof DBH from rat PC 12 cells. Total poly(A)+mRNAfrom dexamethasone-treatedcells was translated in a reticulocyte cell-freesystem. Analiquotwasprecipitated with antisera raised againstTyrOHase.[35S]Methionine-labeledproducts weresep- aratedon aNaDodSO4/10% polyacrylamide gel.Lanes: 1,Coomassie bluestainingpatternofauthentic DBH(25jig);2,immunoprecipita- tionof DBHandTyrOHasewithTyrOHaseantiserafrom totaltrans- latedproductsof PC 12poly(A)+ mRNA;3,totaltranslatedproducts;
4,productsseenwhennomRNA isaddedtothe system. Molecular mass (shown x 10-3)wascalculatedby referencetothemobilities ofstan- dardproteins:phosphorylaseb(92,000), bovineserumalbumin (68,000), ovalbumin(45,000), and carbonicanhydrase(31,000).
wetted with 0.3 M NaCl/2 mM EDTA/0.1% NaDodSO4/10 mMTris, pH7.5/0.1% diethyl pyrocarbonate. After DNA ap- plication, the filterswere bakedfor2 hrat80°C inavacuum oven. Filters were againwetted inthe filterbinding solution andallowed to air dry. Total mRNA prepared from dexameth- asone-treated cellswashybridizedtothe filter-bound DNA in 30%formamide/500 mM NaCl/2mM EDTA/50 mM Pipes, pH7.5/0.5% NaDodSO4. Hybridization continued for 16 hrat 42°C. Nonspecific RNA bound to the filters was removed by extensive washes in 75mM NaCl/7.5mM Na citrate at42°C.
Hybridized RNA was eluted by boiling the filters for 1 minin 300
1,lof sterile H20. Eluted RNAs were precipitated with ethanol, dried, resuspendedin 2,ulofH20, and translatedas described.
Peptide Mapping by Partial Proteolytic Digestion of Au- thentic DBH. The [3S]methionine-labeled protein was syn- thesized invitrofrom mRNA that had been eluted after filter hybridization to a putativeDBH cDNAclone. The radiolabeled protein band was cut out of theNaDodSO4 gel and placedin thesample well of a second NaDodSO4 gel. Partial proteolytic digestion of both the translated protein and of authentic DBH wasperformedasdescribedby Clevelandetal.(21) withS. au- reus V8 protease. Peptide patterns were revealed by the silver stainingprocedure described by Oakley et al. (22) followed by autoradiography.
Totalpoly(A)+ mRNA waspreparedfrom dexamethasone-stim- ulated PC 12 cells and translated invitro in the rabbitretic- ulocytesystem. Translationproductswereimmunoprecipitated with a rabbit serum directed against rat pheochromocytoma TyrOHase.Analysisof the translation pattern indicatedamajor bandat60,000 daltonscorrespondingtothe subunit molecular weight of TyrOHase (3) and a 75,000-dalton protein corre-
1 2 3 4 5 6 7 8
DBH- - 68
FIG. 2. Sucrose gradient fractionation of poly(A)+ mRNA from dexamethasone-treatedPC12cells.Aliquotsofthe indicated fractions weretranslated invitro,and translationproductswereanalyzed by NaDodSO4/10% polyacrylamide electrophoresis. Fractions enrichedfor DBHmRNA(6,7)werepooled and usedtopreparecomplementaryDNA.
Daltonsareshown x 103.
Proc.Natl.Acad.Sci. USA 80
sponding to the subunit molecular weight ofDBH(23) (Fig. 1).
One hundred micrograms ofpoly(A)+ mRNA was then frac- tionated on a 5-20% sucrose gradient. Analiquot of each frac- tionwastranslatedin vitro,and fractions6and 7, enrichedin DBH mRNA activity, werepooled (Fig. 2). Analysis of theop- tical density profile(datanotshown) indicated that these frac- tions weresomewhatlarger than the 18S peak.
MolecularCloning of the cDNA for DBH. Enriched mRNA (15 ug)wastranscribed into cDNA; subsequently double strands wereformed and inserted into the Pst I site of pBR322 by the d(G&C)n-tailing method. Five hundred tetracycline-resistant, ampicillin-sensitive cloneswereobtained.
Transformants were picked onto nitrocellulose filters, grown overnight, and then lysed on gauze pads saturated with 0.1 M NaHCO3/1% Triton X-100/2 mg of lysozyme per ml in an at- mosphere saturated with CHC13. Filters were subsequently placedon gauzepads saturatedwith 0.1 M NaHCO3/0. 1% Tri-
FIG. 3. Detection ofimmunoreactiveclones by colonyimmunoas-
say.Coloniesweregrown onnitrocellulosefiltersand thenlysedinsitu.
Proteinsfrom the lysateswereblottedontoCNBr-activated filterpa- pers.Antigens covalentlyboundtothe CNBrpaperweredetectedby reactionwithTyrOHaseantiserum,followed by125I-labeledproteinA andautoradiography. (A) Colonies (100) wereincubated with Tyr-
OHaseantiserum(1:300). (B) Duplicatecolonieswereincubatedwith antiserumpresorbedwith 500ugof coldTyrOHase;coldTyrOHase (100 ng)wasalsospottednext tothecolonies.(C)ColdTyrOHase (100 ng)
wasspottedontoactivated filters andprocessedwithuntreated Tyr- OHaseantiserum.
ton X-100and then onto filter papersoakedinthesamebuffer.
Activated CNBr paper that had beendampened with binding buffer was placed over the lysed colonies, and antigen binding wascarried out for 4 hr at 40C.
Remainingfunctional groups on the paper were then blocked with 1 M ethanolamine, pH 9.0, for 1 hr at room temperature.
Treatment with 2% glycine as suggested by KempandCowman (16) proved insufficient in our hands to prevent high filter back- grounds after autoradiography. After inactivation, immobilized proteins were allowed to react with pretreated TyrOHaseanti- sera. Without removing antibacterial antibodies, all colonies appeared to give a positive signal. Similarly, the appropriate dilutionof antiserawasnecessarytopreventfilterbackgrounds from obscuring positive signals. A dilution of 1:300 of the pre- sorbedantibody was used in all experiments shown.
Ofthe 500 colonies, 12 gave a positive signal of varying in- tensity(Fig. 3). From these 12 colonies, 3 gave a strong positive signalin eachoffour experiments, 6 had weaker signals in all experiments, and 3 failed to immunoreact in subsequent ex- periments. Allof theimmunoreactiveclones could be displaced by serapresorbedwith native TyrOHase, indicating the reac- tion wasspecific for this enzyme or crossreacting species, or both.
AnalysisofplasmidDNAfrom the nine immunoreactive clones indicated that all nine weredifferentbased on restriction en- donucleasedigestions. Insertsizeof theimmunoreactiveclones ranged from 300basepairs to 3.5kilobases, with an average size of500basepairs. Withthe exception of the 3.5-kilobase transformant, theremaining clones would not be long enough tocode forasubunit of either TyrOHase orDBH. Apparently, even ashort insert sequencecovalentlylinked to the E.coli(3
1 2 3 4 5 6
FIG. 4. Autoradiograms of[35S]methionine-labeledin vitro trans- lationproducts synthesizedwith mRNAcomplementary topDBH-1.
CsCl-purifledplasmid DNAwasdenatured and bound to nitrocellulose filters. Filters werehybridizedwithtotal PC12RNA. RNA bound to thefilterswaseluted thermally and subsequentlyanalyzed by cell-free translation.[3S]Methionine translation products were separated by electrophoresis onNaDodSO4gels and autoradiographed. Lanes: 1, translationproducts of total PC12RNA; 2,products immunoprecipi- tated withTyrOHase antisera; 3, immunoprecipitation of thetrans- latedproduct synthesized with RNA selected by pDBH-1; 4, total trans- lationproducts; 5, total translation products whennoRNA wasadded tothe system; 6, authentic DBH (25pg)stainedwithCoomassie blue.
2164 Biochemistry: O'Malleyetal.
lactamase geneproducesenough of the fused polypeptide to be detected, atleastby a polyvalent antibody.
FilterHybridization and Translation. The plasmids from the nineimmunoreactiveclones wereindividuallypurifiedand used toselect complementaryRNA inthe filterhybridization-posi- tiveselectionprocedure. SelectedRNAswere translated in vi- tro inthe rabbitreticulocyte system;translated productswere displayed on NaDodSO4 polyacrylamide gels. From the nine clones,RNAselectedfrom one revealed a uniquebandof 75,000 daltons (Fig. 4). This protein whichcould be immunoprecipi- tated with TyrOHase antiseracomigratedwithauthenticDBH.
Thisplasmidcontained an insert of 330 base pairsandwasdes- ignatedpDBH-1.
Peptide Mapping of the Protein EncodedbypDBH-1 and Native DBH. Inorder tounequivocallyidentify this clone as containingnucleotide sequencesspecific forDBH, weanalyzed peptidepatternsobtained from theproteinencodedbypDBH- 1.The[35S]methionine-labeledproteinobtained fromthe pos- itiveselection procedurewasremoved fromthe gel and sub- jected to partial proteolysis with S. aureus V8 protease. Si- multaneouslywedigestedasampleof authenticDBHthat had similarlybeen cut out of aNaDodSO4 gel. Digestionproducts weredisplayed on 15% NaDodSO4gels thathad been stained with silver. Afterautoradiography, methionine-labeled bands werecompared withthosegeneratedbythe partialproteolysis ofthe authentic protein. As seeninFig. 5, thelabeledpattern corresponded with that of the authentic DBH. Toverify this result, gelslicesfrom thetranslatedproteinandauthenticDBH were incubated also with
"Iin ordertolabel all of the pep- tides, notjustthose containing methionine groups. Again, in this experiment, all of the labeled bands observed inthe au- thentic DBH peptide pattern matched those ofthe plasmid- specificprotein. Inthe latter case, additional bandsalsowere observed, presumably
correspondingto acomigratingreticu- locytelysate protein(datanotshown).
1 2 3 4 5
FIG. 5. Peptide mapping ofauthentic DBH and the translated product obtainedfrom RNA selected by pDBH-1. Peptidemappingwas
done withgel slices obtained from theelectrophoresis of eitherau-
thentic DBH(25,ug)orthe translatedproduct fromRNAselected by pDBH-1. Sampleswereincubated with1,ug ofV8proteaseina4-cm stacking gel priortoresolution ina15%NaDodSO4/polyacrylamide gel.Oneexperiment (lanes1-3)showsthe silver-stained peptidepat- ternsfromonegel slice ofauthentic DBH (lane 1) andonegel slice ofthehybrid-selected translation product (lane2);lane3shows the [35S]methioninepatternoflane 2 after 2 wk ofautoradiographicexpo- sure.Inanotherexperiment(lanes4and 5), five gel slices ofthe hybrid- selectedtranslationproduct andonegelslice of the authentic DBHwere incubated with5,ug of V8protease.Byincreasing theamountof the translated product ofpDBH-1, silverstainingalonewassufficientto demonstrate thesimilarityinbandingpatternsofauthentic DBH(lane 5)andthehybrid-selected product (lane 4).
Whatappear tobe shared antigenic sites of thecatecholamine pathwayenzymesenabledus toobtainfromPC 12cellsa mes- senger RNA fraction enriched for DBH mRNA by using TyrOHase antiserum. The enrichedDBH mRNAwasthen used toprepare acomplementary DNAlibrary from which aDBH cDNAclonewasisolated. Inthe absence ofareportedamino acidsequencefor DBH, thefollowingcriteria wereusedto es- tablish the identity of the DBHclone. (i) Cloneswerepicked that synthesized peptides crossreacting with TyrOHase anti- serum. Fusedpolypeptides from expressingtransformantscould bedisplaced byusing antiserumpresorbed with nativeTyrOHase.
(ii) One such clone selected a mRNA that, when translated in vitro, formed a protein of 75,000daltons, thesizeofDBH. (iii) The translated protein could be immunoprecipitated with TyrOHase antiserum andcomigrated with authentic DBH. (iv) Partial proteolysisof both authentic DBH and the protein en- coded by pDBH-1 produced identical peptide patterns.
The results described here demonstrate the effectiveness of thecolony immunoassayprocedure. Ageneraldisadvantageof the method is that only one in six clones would contain an insert in the rightorientation andreadingframe tocode forafused polypeptide. However, enriching for the desired messenger population,inserting double-strandedcDNA asclosetothe full length aspossible, and screening additional transformants would increasethechances ofdetectinganexpressing transformant.
Kemp and Cowman(16) detectedevengreater proportions of positive transformants whenfull-length inserts were screened bycolonyimmunoassay,probablyduetopeptideinitiationwithin the insert itself(24, 25). But as demonstrated in the case of pDBH-1, even small inserts can generate a fusedpeptide car- rying immunoreactive determinants.
Aswith all immunologicalprocedures, antibody specificity iscrucial. Of the othereightimmunoreactiveclones described here, two provedtobe clones ofanoncatecholamine-related proteinslightly recognized bythe TyrOHaseantibody. Affinity- purified antibodies should increase sensitivity further and min- imizeselection of false positives.
This raises anotherissue. Although the preliminarydata of
Johetal.(8) suggests that the catecholamine biosyntheticen- zymes, TyrOHase, DBH, and PhEtnMeTase share antigenic determinants, it isalsopossiblethat the antigensused to gen- erateantiseraarecross-contaminatedbecause the antigensare derived from tissues inwhich all three enzymesarepresent.
Theavailability ofa rat cDNAprobe forDBHwillallowin- vestigation ofthetranscriptional regulation of the DBH gene inthe PC 12cell line. The clonal cell line notonlypossesses thehighly differentiatedproperties oftypicaladrenergiccells but alsoexhibits responses typicalof
earlycells inthe neural crest(5,6).Assuch, thePC12cell lineshould beagood model for additional studies examining mechanisms responsible for differentiation of the neuralcrest invivo.
Recently, it has been reported that rat cDNA clone for TyrOHasecross-hybridizedwithmRNAfrom human tissue(26).
Presumably,thisalso will betruefor DBH,sothat the rat cDNA clonecould be used to
identifycorrespondingsequences inclones fromahuman genomic DNAlibrary.Then human probes could be used to investigate atypical catecholamine function at the molecular levelin humans.
Wethank George Makk forexcellent technical assistance. This work wassupportedinpartbyNational Institute of Mental Health Grant MH 23861andOffice ofNaval Research Grant SRO-001 to J. D. B., and by grantsfromthe National Institutes of Health and Veterans Administra- tionto L.K.
Proc.Natl. Acad. Sci. USA80
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