cDNA Sequences Reveal mRNAs for Two G{alpha} Signal Transducing Proteins from Larval Cilia

10 Pages·1991·4.5 MB·English

by  L M Wodicka

Reference: Biol. Bid! 180: 318-327. (April, 1991)
cDNA Sequences Reveal mRNAs for Two Ga Signal
Transducing Proteins from Larval Cilia
LISA M. WODICKA AND DANIEL E. MORSE
Department of BiologicalSciences andthe Marine Biotechnology Center,
University ofCalifornia, Santa Barbara, California 93106
AHbstract. In planktonic larvaeofthegastropod mollusk, shown in other systems to control the activity of phos-
allotis nifescens(red abalone), settlement behaviorand pholipase C; the other is more closely related to G, and
subsequent metamorphosis are controlled by two con- G . These results extend the tractability ofthe Haliotis
vergent chemosensory pathways that report unique pep- system to analyses of cDNA and protein sequences of
tide and amino acid signals from the environment. The chemosensory elements from isolated cilia. This is the
integration of signals from these two sensory pathways firsttimethat mRNA hasbeen purified from isolatedcilia,
providesforvariableamplification, orfine-tuning,oflarval and the corresponding cDNA synthesized and character-
responsiveness to the inducers of settlement and meta- ized.
morphosis. Thesepathwaysmaybeanalogous tothe neu-
ronaland molecularmechanismsoffacilitation and long- Introduction
term potentiation characterized in other(adult) molluscan
systems. Recently, thechemosensory receptorsandsignal Larvaeofthegastropod mollusk, Haliotisnifescens(red
transducers apparently belonging to the regulatory path- abalone), undergo a dramatic behavioral change when
G
way (including a protein and protein kinase C) have they encounter a specific chemical cue at the surfaces of
been identified in cilia purified from H. nifescens larvae. crustose redalgae: the planktoniclarvaeceaseswimming,
These elements retain theirsequential receptor-dependent attach to thealgal surface, andcommence metamorphosis
regulation intheisolatedcilia in vitro. Asafirststeptoward and plantigrade locomotion and feeding. Thisbehavioral
the molecular genetic dissection ofthe receptors, trans- transition is controlled by the integration oftwo conver-
ducers,andthe mechanismsoftheircontrol ofsettlement gent chemosensory pathways that respond to chemical
behavior and metamorphosis, we present evidence that signals from the environment: a morphogeneticpathway
thecilia purified from these larvae contain polyadenylated activated by a GABA-mimetic morphogen encountered
mRNA
corresponding to unique signal transducers. Pu- by the larvae on surfaces ofrecruiting algae, and a regu-
rification ofthis mRNA, enzymatic synthesis ofthe cor- latory or amplifier pathway stimulated by lysine in sea-
respondingcDNAs,amplification bythepolymerasechain water(Morse etal., 1984; Trapido-Rosenthal and Morse,
reaction, cloning, and sequence analysis reveal that the 1985, 1986a, b; Baxter and Morse, 1987; Morse 1990a).
mRNA
ciliary includes sequences that apparently code Activation of the morphogenetic pathway receptors is
for two Ga signal transducing proteins. One ofthese is thought to trigger an efflux ofchloride or other anions
highlyhomologoustomembersoftheGq family, recently across the membrane ofthe primary chemosensory cell,
apparently resulting in excitatory depolarization (Baloun
and Morse. 1984). This transduction of the exogenous
Received 9July 1990;accepted 28January 1991. chemical signal toonethatcan bepropagatedbythelarval
1 Abbreviations: GITC. guanidinium isothiocyanate: SDS. sodium nervoussystem isevidently sufficientto inducethechange
dimnoeddtoehlcyyylllgaasmluialfcnatotomes;eidtIehP:aTnAGe.M;Vis,EopDarTvoipAya.ln-fmeMythehilyoolgbealnlaeasdctitaoomssiiinsdeev-;itreXut-srg;aaTl-r,aicsbe,rtotimrcios-a-,chiycddh;rlooTxrEyo,-- ainndlatrhvealstbaerhtaovfimoertcaumlomripnhaotsiinsg.iAnctsietvtalteimoennotf,tahtetaamcphlmiefnite,r
Tris-EDTA;TBE,Tris-borate-EDTA;TAE.Tris-acetate-EDTA;bp,base- pathway receptors increases the sensitivity or output of
pair(s). Thestandard one-letteraminoacidcode isused. the morphogenetic pathway by as much as 100-fold. The
318
Ga CDNAS FROM LARVAL CILIA 319
receptorsoftheamplifierpathwayareactivated when they grated to control behavior in these small larvae (Morse,
bind lysine, lysine polymers, or certain lysine analogs 1990a). As a first step toward that objective, we report
(Trapido-Rosenthal and Morse, 1985. 1986b). Experi- here the amplification, cloning, and partial sequence
ments //; vivodemonstrated that the amplifierpathway is analysis ofcDNAs apparently corresponding to two Ga
mRNA
controlled by chemosensory receptors and signal trans- signal transducing proteins, from purified from
ducersdistinct from thoseofthemorphogeneticpathway, the isolated cilia.
and that the lysine receptors ofthe amplifier pathway ac-
G
tivate a sequential protein-(phospholipase C) diacyl- Materials and Methods
glycerol-protein kinase C signal transduction cascade
(Baxter and Morse, 1987). This system ofdual control, Cilia isolation
in which the integration of two different kinds of che- Larvae ofHaliotis rufescenswere produced in the lab-
mosensory signals from the environment modulates the oratory by hydrogen peroxide-induced spawningofgravid
settlement behavioroftheHaliotislarvae, fine-tuneslarval adults (Morse et ai. 1977). Larvae were maintained at
responsiveness to exogenous settlement cues. The result 15C in 5 /urn-filtered, UV-sterilized running seawater
ofthisintegration mayenhancethesite-specificityoflarval until 7 days post-fertilization; at this time they become
settlement and metamorphosis in potentially favorable developmental^ competent to metamorphose in response
habitats (Trapido-Rosenthal and Morse, 1985, 1986b: to inducer (Morse et ai. 1979, 1980). Cilia were purified
Morse, 1990a, b). by differential centrifugation, after abscision induced by
Because both the morphogenetic and amplifier path- exposure ofthe larvae to a mild calcium-ethanol shock
ways can be activated by macromolecular (protein-asso- (Baxter and Morse, in prep.). This method is a modifi-
ciated or polypeptide) ligands that are presumably im- cation ofthat used for the purification of functional re-
permeant (Morse ct a/.. 1984; Trapido-Rosenthal and ceptor-bearing cilia from olfactory epithelia (Rhein and
Morse. 1985. 1986b), it was suspected that the chemo- Cagan, 1980; Chen and Lancet, 1984) and other sources
sensory receptors controlling these two pathways might (Watson and Hopkins, 1962; Linck, 1973). Electron mi-
be located on externally accessible epithelia (Morse, 1985, crograph examination revealsthe purifiedciliatobeintact
1990a, b). Epithelial cilia are known to carry chemosen- and completely free ofcell bodiesanddebris; theciliaare
sory receptors in a wide variety ofsystems, including the heterogeneous, and include short (ca. 0.5 ^m) spatulate
well-characterized olfactory epithelia of frogs, fish, and ciliasimilartothesensorycilia found in otherinvertebrate
mammals(e.g., Rhein andCagan, 1980; Chen and Lancet systems, and long(>10 ^m) propulsivecilia from the lar-
1984; Pace el at., 1985; Pace and Lancet, 1986; Lancet val velum (Baxter and Morse, in prep.).
and Pace, 1987; Anholt, 1987; Anholt el ai. 1987). Epi-
thelial cilia also have long been suspected to carry the RNA
isolation
chemosensory structures that mediate substratum rec-
ognition and thereby control settlement behavior and We isolated total RNA from cilia freshly purified from
metamorphosis in variousmolluscan larvae(Raven, 1958: Haliotis rufescens larvae, using a single-step extraction
Fretter and Graham, 1962; Bonar, 1978a, b; Chia and with an acidguanidinium isothiocyanate(GITC1(-phenol-
Ross. 1984; Yool, 1985). chloroform mixture (Chomczynski and Sacchi, 1987).
Recently, epithelial cilia isolated from H. rufescenslar- Poly A+ mRNA was purified using oligo (dT) cellulose
vaewere shown to contain the lysine receptors and signal columns eithercentrifuged (Clontech, Palo Alto, Califor-
transducers that may control the amplifier pathway in nia) or used with a syringe (Stratagene, La Jolla, Califor-
vivo (Baxter and Morse, in prep.). These elements retain nia) in a modification ofthetechnique described byAviv
their functional coupling in the isolated cilia in vitro: i.e., and Leder(1972).
the specific and saturable binding of lysine to sodium- RNAwaspurified from bovineretinatoprovidea pos-
independent lysine receptors activates sequentially a G itive control enriched forG protein (transducin) mRNA.
protein and diacylglycerol-stimulated protein kinase C For this purpose, fresh bovine eyes were obtained from
(Baxter, 1991; Baxter and Morse, in prep.). The lysine- Federal Meat Market (Vernon, California). Retinas were
binding receptor was found to be reciprocally regulated immediately dissected on ice in Tris-buffered saline
byitstightlycoupledG protein inthecilia in vitro(Baxter (Maniatisetai, 1982) and placed on dry icefortransport
and Morse, in prep.);similarbehaviorisexhibitedbyother toanearbylaboratory. There, halfthesampleswerefrozen
members ofthe rhodopsin and /3-adrenergic G protein- in liquid nitrogen, and halfwere homogenized in GITC;
coupled transmembrane receptor superfamily. these samples then were transported to Santa Barbara in
The tools ofmoleculargenetics are required to further GITC or on dry ice. RNA was isolated by the GITC-
resolve the mechanisms by which the chemosensory and cesium chloride centrifugation method (Chirgwin et ai,
mRNA
neuronal receptors, transducers, and pathways are inte- 1979), and was then purified asdescribed above.
320 L M. WODICRA AND D E. MORSE
Total RNA from both sources was analyzed by elec- After synthesis, oligonucleotides were deprotected at
trophoresis on formaldehyde gels (Maniatis el at. 1982) 55C in ammonia overnight; theywerethen purified and
with ethidium bromide added to the sample buffer, or detritylated by reverse-phase chromatography (Oligonu-
wasdenatured at 65 C for 15 min. quickly chilled on ice. cleotide Purification Cartridgesfrom Applied Biosystems).
electrophoresedon 1% agarose/TBEgels(Han elat. 1987) The oligonucleotides were dried down, resuspended in
and visualizedby UV-excited fluorescenceafterethidium sterile water, and concentration was estimated by absor-
bromide staining (Maniatis el at. 1982). Purity oftotal bance at 260 nm.
and poly A+ RNA was confirmed by the ratio ofabsor-
bances at 260 and 280 nm and concentrations estimated PCR amplification
from the A2W> For amplification of cDNA, 50 pmol ofeach primer
Synthesis ofcDNA andoligonucleotideprimers aanddde4d0d%ir(e2ct0l^yDtoof10t0hen\rePveCrRserteraacntsiconrsi.ptAilolnortehaecrtiroenacwteiroen
Reverse transcriptase from avian myeloblastosis virus components, including Taq DNA polymerase, were pur-
(fmAirRsMtNVs)Atra((In1ndvMicgt)DroNwgAae.sn.uCSsialenidaDfimoerRgeoNa,cAChA(5)10w0ja0uls-5rue0sa0ecdtnigtoo)n.soyrntbhoevsiinzee cnnehucamtsibecedurt)forafonmadmpPulesirefkdiicanastEsiluogmngeecrsy-tcCeledetsbuwysatsChoavrtaprm.iaen(duNfofarrcwotamulrk2e,5r.tCoTohn4e-5
For polymerase chain reaction (PCR) amplifications, with the following parameters: denaturation at 94C, 1
two kinds ofoligonucleotide primers were made: degen- min;annealingat 37C, 1 min; extension at 72C. 3 min.
erate primers(D) were used forthe first amplifiGcaaticoDnsNoAf Foramplification ofgenomic DNA usingspecific primers,
thecDNA, and(oncetheexactsequenceofthe 0.3 Mg Haliolis rufescens sperm DNA. 0.75 ng Telrahy-
wasdetermined)specific primers(S)wereusedtoamplify mena ihermophila DNA, 0.5 Mg Vibrio harveyi DNA, or
ttihdeesgeunsoemdiacssperqiumeenrcsesfofrrPomCRspweerrmeDsNynAt.hesOilziegdon(uacsletoh-e a1mMpglisfailcmatoinonspreeracmtiDonNsA. (wTehreeHaadldioeldist.ooTtehlerrawliisyemeindaen.tiacnadl
trityl-derivatives) by an automated oligonucleotide syn- I'ibrio genomic DNA samples were generously provided
thesizer(Applied Biosystems Inc., FosterCity, California). by Jay Groppe. Jennifer Ortiz, and Richard Showalter,
To reduce the degeneracy ofthe primers, Haliolis rufes- respectively.) All DNA samples were tested for amplifi-
censcondon usage frequencies(Groppe and Morse, 1989) cation at amounts equal to or greater than the number
were taken into consideration, and two separate pools of ofgenomeequivalentsoftheHaliolisDNA. BecauseDNA
the downstream primer were synthesized (D2 and D,). samples were in TE buffer, additions were adjusted such
Degenerate oligonucleotide primer sequences corre- that the total amount ofTE (and thus the concentration
sponding to the conserved G and G' domains (Lochrie of EDTA) in all PCR reactions was equal. Optimum
and Simon, 1988) of G protein a subunits are: D,: 5' magnesium concentration, primerconcentration,andcy-
GAAGGATCCAAGTGGATCCA(GC)TG(CT)TTT
3': cling parameters were determined empirically. For am-
(D,A:G)5'AACT3'C:ADA3G:C5'TTCTTCCCATA(GTCGT)TCTTCTT(TA(GT)GT)TC(TTTG()AAGG)-- pplriifmiecartiwoansofusgede;notmheicfiDnaNlAc,onc2e5ntprmatoilonofofeamcahgnsepesciifuimc
TG'T(dComAa)iAnG(aAmiGn)oAAaci3'd. Ds,equceonrcree,spKonWdIs(tHoQt)hCeFc;onDse2ravnedd awatsotailncorfe3a0seadmpflriofmicatthieosntcayncdlaersdwa1s.5pmerAf/ortmoe2dmasAIbe:foarned,
D, correspond to the conserved G domain sequence except that theannealingtemperature wasraisedto45C
FLNK(KQ)D. Amino acid sequences chosen for these for the 2nd cycle and to 55C for the 3rd-30th cycles. A
domains were based on the findings ofStrathmann el at 5 min extension step (72C)wasaddedafterthelastcycle.
(1989), with inclusion of a degeneracy representing ad-
ditional sequencesdetermined foryeast. In addition, these Gel electrophoresisforanalysis andpurification ofPCR
purndiemrelrisniinngc)lucdoerroelsipgoonnudcilnegottoidteheseBqaumenHceIs(D(,in)daicnadteHdinbdy reactionproducts
wIIeIr(eDa2dadneddDa,s)lirenskterriscttioofnaecinlzityamteectlaorngeitnsg;otfhetsheesaemqpuleinfcieeds onPaCgaRrorseeactgeilosn(pr3o%duNcutssewievreeaagnaarloyszeedplbuysel1e%ctSreoaphpolraeqsuies
products. AshortervariantofD, alsowasproducedwith- agarose in TBE for cDNA-PCR reactions and 1.5% aga-
out the 9-nucleotide linker. Specific (non-degenerate) rose/TAE bufferforgenomicreactions), runat2-6 v/cm.
primer sequences based on the cDNA sequence subse- and stained with ethidium bromide. For analysis on the
quently determined forthe HaliolisciliaGl (seebelow) higher percentage gels, samples were loaded into wells
a3'r;e: Sa,n:d5' GSC2:AG5G' ATCCTCCAACAGGTCCTCTACTGCGAGTTGATGTGCTTGTAA- cdaisgtesfterdompl0a.s7m%idag(aProBsRe3.22O-nBest^Ng o1.ffrersotmricNteiown eEnnzgylmaend-
TAATCGT 3'. Theseprimersalsohave9-nucleotide long Biolabs Inc., Beverly, Massachusetts) was included for
5'-linkers with a BamH 1 site (S,) or a Hind III site (S2). molecular weight markers.
G cDNAs FROM LARVAL CILIA 321
Forpurification, amplified cDNA waselectrophoresed resulting sequences analyzed with the aid ofthe Pustell
on 3% low melting temperature agarose (Mermaid, from Sequence Analysis software (IBI Macintosh).
DNA
Bio 101 Corp., La Jolla), and bands were excised
while visualized on a 365 nm light box. DNA then was Results
removed from theagarose bybindingtoglassbeads(Glass
Fog, from Bio 101 Corp.). Genomic products were run PCR amplification ofcilia Ga cDNA
on 1.5% agarose gels as described above; bands were ex-
cised, and DNA was purified by binding to glass beads Poly A+ mRNA was isolated from the purified cilia of
(Geneclean, from Bio 101 Corp.). 7-9-day-old, competent larvae of Haliotis rufescens. as
described in the Methods. Startingwith about 10" larvae,
DNA cloning typical yields were 300-400 mg (wet weight) cilia, 50 ng
Purified PCR products and a recombinant plasmid tRoNtaAl).RNAAM,Vanrdeve1r^seg torfanpsoclryipAta+semwRaNsAuse(d=2t%o coaftatloytzael
dvfieogcletlsootrweed(d/7abBtylu3eB7scaCrmipHftoIrK.(1SeIhnIz+i,ynmfe2rs0omnf\rSovtmoralNtuaemgewesneEwniCgtolhrapn.H)dinwdBelirlole- drfuraponlmdeotxmhe-whapuserxitafhimeeendrumspRerNdiAmaes,dtaesnmydnptlthaheteseirsefsoourfltPfiiCnrgsRtmsaRtmrpNalAnidf-icccaDDtNNioAAn
labs). Digested products were purified by gel electropho- with the degenerate primers corresponding to the con-
resis as described above. The purified, linearized vector served G and G' domains, as described in the Methods.
ab( y1m0o0Tu4nngt)DotNfhAPenClRwiagpassreolidoguavcteterdninitsgoehrattn;atathpips4rCroe.xaictmCiaootnnetrlwoyalsesqcauwtiiatmlhoylznaeord ath1eT9hl6aerbvppalrpircmiolediruasc(tFuisfge.rdolmca)lt.ehaTerlhcyeDdsNiirzAeectoteefmdtphtlihasetpearsmoppdlruiecfptiacriasetdwiioftnrhoiomnf
added insert were treated identically. Transformation of the range predicted foraG cDNAdomain lyingbetween
recipient bacteria (Epicurian Coli XL-1 Blue, from Stra- the highly conserved G and G' domains. This result sug-
(t1a9g8e3n)e,Cwoirtph.)mwoadisfipceartfioornmsedrebcyotmhmeemnedtehdodbyofStHraantaagheanne gests that themcilRiaNpAurified from HaGliotis rufescenslarvae
may contain coding for a protein.
Corp. After transformation, colonies with recombinant The positive result shown in Figure la allowed us to
plasmids were identified on the antibiotic-containing agar furtheroptimizethe primers used forPCR-amplification.
medium with the chromogenetic substrate, X-gal. Each Theupstream primerused inthefirst PCRamplifications
clone wasthen subcultured in 5 ml ofLB containingDaNmA- wasrelatively short, consistingofadegenerate poolof17-
picillin and tetracycline (37C, overnight). Plasmid mers. Thisprimeronlyweaklyamplifiedthepositivecon-
was purified from these cultures after lysis with alkalai, trol template, cDNA from bovine retina, a tissue highly
usiCnlgontheedmpilnaismpirdepDpNrAocwedausrdeig(eMsatneidaatsisabeolvael.w,it1h98B2)a.mH sehnorwinch)e.dThfoeratdhdeitGion okfnoniwnne nausclteroatnisddeuscicnon(traeisnulitnsgntohet
IIIIaa(nnfddorHHwiihnniddchIIIItIIhsesiitpemlsu)al.stmRaiendsetohruaiscsltyti,wonoadnsiidtgeessDs,etNpfsalAwraaentrkeielnyganwtahiletyhBzeBadsmsbHHy tsMhaeetqeu5re'in-aeclnesdoaofnfdtthhMeeetB1h7ao-mdmseHr)Is,.rmteasoktgreiescnteeirfoafnticeeinetndhtoenaDum,pcllpierfaiiscmeaetrsisiotne(stoeoef
agarosegelelectrophoresis. Plasmid waspurified by the control Ga sequence from bovine retina cDNA pos-
cpernetpriSfpuugantiCoonlucmhnrso,maftroogmraPphhayrm(aSceipah,acPriyslcaSt-a4w0a0y,MNineiw- sible (Fig. Ib). [Similarly, we had found earlier that ad-
dition ofthe nine nucleotides containing the sequence of
Jersey). the Hind III restriction site to the 5'-end ofshort down-
D
DNA stream primers, to generate the 26-mer 2 and D, pools,
sequence analysis also significantly enhanced the efficiency ofthese oligo-
Di-deoxy sequencing reactions were performed with nucleotides as primers. These non-matching nucleotides
modified T7 DNA polymerase (Sequenase II; United donotreduceprimerspecificity. Similarobservationshave
States Biochemical, Cleveland, Ohio) and 5' [-35S]dATP, been reported by others (e.g.. Mack and Sninsky, 1988).]
1 100 Ci/mmol (Amersham) by procedures modified from The results in Figure Ib show the downstream primers
Sanger el al. (1977). Primers used for sequencing were in the degenerate pool D; are more effective than those
plasmid primers T7, T3, KS, and SK (purchased from in D3 for detecting and amplifying Go cDNA sequences
aSbtroavtea.geRneea)ctainodnsthweersepeacinfailcypzreidmbeyrselSe,ctarnodphSor:e,sdiesscornib8e%d fPrroimmebrotDh,bdoifvfienres frertoimnaDan:dbyHatlwiootniuscrluefoetsicdeenssalanrdvaalpcpialira-.
G
polyacrylamide-50% urea wedge sequencing gels. Gels ently fails to hybridize efficiently to the target protein
werewashed in 10%aceticacid-10% methanol,driedwith cDNA sequence. Agarose gel electrophoresis of PCR
vacuum at 80C, and subjected to autoradiography. The products amplified with the optimized 26-mer primers
D
autoradiograms were read with a sonic digitizer, and the (D, and 2) reveals the expected 205 bp product from
322 L. M. WODICKA AND D. E. MORSE
cDNAs from both the larval cilia and bovine retina. The DNA (S) A. rRet. -Cilia- nNone
product is nine nucleotides longerthan that seen in Figure 2nd Primer - (X) D2 D3 D2 D2 D2 D3 D2 D2
la, asexpected because the upstream primer(D, ) is nine Cycles - 30 30 30 30 35 40 40 45 45
nucleotides longer than that used in the first experiment.
The cilia cDNA required more cycles of amplification
(35) than did the bovine retina cDNA (25) before the
product on an ethidium bromide stained gel could be
visualized. Thus, there may be greater primer-template
mRNA
mismatch, orthetarget may be less abundant, in
the larval cilia.
A control PCR reaction with noadded DNA template,
atest for DNA contamination ofreagents, yielded no de-
tectable PCR products amplified after45 cycles(Fig. Ib).
121
In addition, no amplified PCR products were observed
in the following control reactions (not shown): (a) no
primers in the PCR reaction; (b) no Taq polymerase in
the PCR reaction; and (c) no reverse transcriptase in the
cDNA
reaction.
9 10
Cloning andsequence analysis ofGa cDNA
The 205 bp PCR product from the cilia cDNA was Figure l(b). PCRamplificationoflarvalciliacDNAusingdegenerate
cloned in the plasmid vector as described in Materials primers D, andeitherD,orD,. Lanes: (1)molecularweightstandards;
and Methods. Twelve transformant colonies were picked (2)\DNAandprimersasPCRcontrol;(3)bovineretinacDNA.primers
zaynmde-sdubicguelsttuerdedp;lgaeslmeildecDtrNopAhorsehsoisweodftthheatres1tr1icotfiotnheens-e cpDyr|cilmaeesn;rds(D5D),2c.ia3lni0adccyDcD,lN,esA;3,5(4pc)ryciblmoeevsri;sn(e7D),rectiailninadaccDDD,N,NAA3,,0pcpryrciilmmeeesrr:ss(6DD),,caialnnidda cDDD23N,,A43,00
cloSneeqsuceonncteaianneadlyisnissertosfotfhrtehee coofrrtehcet csilzoen.ed cilia PCR cpyrcilmees;rs(8D),cialniadcDD2N,A4,5 cpyrcilmees;rs(1D0,) naondDDN3A, 4t0empclycaltees,(p9r)imcielrisa cD,DNaAn.d
products revealed two unique cDNA sequences (Fig. 2). D:, 45 cycles.
As shown, both of these share a number of the highly
conserved residues ofother G proteins in the G-G' re-
gion. Two outofthethreeclones provedtohaveidentical nucleotide (and deduced protein) sequences over the 51
G
amino acid region between the primers. This Haliotis
Cilia protein subunit (Gl) differs in the region analyzed by
only one amino acid from the sequence ofmouse brain
G 1 1, a member ofthe newly discovered Gq class ofa
subunits (Strathmann el ai, 1989; Strathmann and Si-
mon, 1990). This sequence differs significantly in the re-
gion analyzed from all other known classes ofG protein
196bp aDrossuobpuhniiltas,(aGnsd, Gy,e,astG. Tn,heGs,ecGo,,ndGGJpfrrotoeminmasmeqmuaelnsc.e
from the larval cilia (Haliotis G2) is most homologous
to G and GJ from Drosophila,
Amplification, cloning, andsequence analysis ojGot
DNA
from Haliotis genomic
The nucleotide sequence from the Haliotis ntfescens
larval cilia Gal clone was used to design specific (i.e..
non-degenerate) primerstoamplifythecorrespondingre-
PCFRi.guPrreodlu(ca)t.frGomp5r0oteciycnleassoufbaumnpiltifcicDatNiAon;frpormimecirlsia,wearmeplaif1ie7d-mbeyr gspieocnifoifcGprimferrosm(HS.} arnudfesSc:e)nwsesrpeebramsegdenoonmircegDioNnAs.ofTthhee
variant ofD, (without the BamHI site) and D2. Number ofbase-pairs GHaliotis sequence that differed significantly from other
in product isindicated. Details in Materialsand Methods. protein sequences (Fig. 5; cf. Fig. 2).
Ga cDNAs FROM LARVAL CILIA 323
Inlron Ident/51
Gal Ga2
HaliotisGal ENVTSIMFLVALSEYDQVLVESDSENRMEESKALFRTII TYPWFQNSSVIL 26
HaliotisGtt2 EGVTAI 1 FI VAMSEYDLTLAEDQEMNRMMESMKLFDS ICNMCWFTDTSI 1L 26
MouseGq ENVTS1MFLVALSEYDQVLVESDNENRMEESKALFRTI I TYPWFQNSSVIL SO 26
Drosoph.Gj EGVTAI 1 FCVALSGYDLVLAEDEEMNRMIESLKLFDS ICNSKWFVETSI IL 27 41
Drosoph.G EDVTAI 1 FCVAMSEYDQVLHEDETTNRMQESLKLFDS ICNNKWFTDTSi 1L 28 41
RatG EDVTAI I FCVALSGYDQVLHE0ETTNRMHESLMLFDS rCNNKFF I DTSI IL 27 36
RatG EGVTAI IFCVELSGYDLKLYE0NQT SRMAESLRLFDS ICNNNWFI NTSLIL 25 33
x
Bov.Transd. EGVTC1 I FI AALSAYDMVLVE0DEVNRMHESLHLFNS ICNHRYFATTSIVL 26 32
YeastGP1 EGITAVLFVLAMSEYDQMLFEDERVNRMHESIMLFDTLLNSKWFKDTPFIL 23 30
YeastGP2 DNVTLV I FCVS LSEYDQTLMEDKNQNR FQESLVLFDN IVNS RWFARTSVVL 25 27
Drosoph.Gs NDVTAI I FVTACSSYNMVLREDPTQNRLRESLDLFKS IWNNRWLRT I S I IL 21 27
RatG NDVTAI IYVAACSSYNMVI REDNNTNRL RESLDLFES IWNNRWLRT I S I I L 18 25
olf :::.::::::::-:: ::::::::.:....-..-.
Figure 2. Ga sequences from cilia. Deduced amino acid sequences ofthe cloned PCR products(Gal
and G2) from Haliolis rufescens cilia cDNA are compared with the corresponding regions ofother Ga
subunits. Residues highly homologous in several Ga proteins are indicated by shading. Region shown is
Gbeatlweaennd(Gnoat2in(colfu5di1ntgo)tadle)giesnesrhaotwenprfoirmeeraschD,G.andSeDqu;e.nTcheesnusuemdbetordoefsiagmnisnpoecaifciicdspriidemnetriscaSl,taonHdalSi2otairse
shown byarrows:position oftheintron ingenomic DNA isindicated. Standard(IUPAC)one-letteramino
acidcode isused.
Two distinct DNA products were seen when Haliotis S: as primers for the sequencing reaction. While this
genomic DNA was amplified by PCR reactions with the method ofdirect sequencing proved useful in this case,
S, and S: primers, and the productswere analyzed on an thequality ofthesequencing reactionswas highly variable
agarose gel (Fig. 3). The specificity of these primers for (t/McCabe, 1989).
fHiaclaitoitoinsoDfNgeAnoimsiecviDdeNntAbsyetqhueeinrcfeasilfurreomtoTdeitrreacthyammepnlai,- theThGelgecnoDmNiAc PseCqRuepnrcoedufcrtosmeqtuheenccielisa,wearneditdoenotnicealant-o
Vibrio, or salmon sperm in otherwise identical PCR re- other, in the putative coding regions. But both of the
actions. cloned genomic sequences contain an intron that inter-
The 1.25 kb and 1.45 kb Haliolisgenomic PCR prod- rupts the coding sequence. The exact position ofthis in-
uctswereelectrophoretically purified, separatelyamplified tron between exons 6 and 7 is conserved in the genomic
again, and analyzed electrophoretically (Fig. 4). The suc- sequences corresponding to the HaliotisGal and the G
cessful purification ofthe two genomic PCR products is and G, ofmammals and Drosophila (Figs. 2, 5). The two
shown by the lack ofvisible contamination afterthissec- Haliotis genomic sequences prove to be highly homolo-
ond round ofamplification and gel electrophoresis. Each gous to one another at both ends ofthe introns that are
purified PCR productwasclonedasdescribed above, and adjacent to the coding regions (corresponding to exons 6
the cloned inserts were then sequenced using primers and 7 inotherspecies;cf. Fig. 5);however,thetwointrons
matched to the vector. differ in length by about 200 bp.
During the cloning step we discovered that the larger
ofthe two genomic PCR products had an internal Hind Discussion
III site not found in the smaller product. This difference
apparently resides in an intron, a non-coding sequence The larvae of Haliotis rufescens provide a uniquely
not present in the cDNA (see below). Although the re- tractable model system for resolving and analyzing the
sulting Hind Ill-Hind III fragment was not cloned, a par- chemosensory receptors and signal transducers, and the
tial sequence forthis region was obtained from the direct mechanisms of their functional integration, controlling
sequencing ofthe uncloned PCR products using S, and behavioranddevelopment in responsetochemical signals
324 L. M. WODICKA AND D. E. MORSE
bp
bp
1857
1857
1450
1250
1450 1060
1250 929
1060
929
7 Figure4. Halioliagenomic PCRproductsaftergel purification and
30 additional cycles ofamplification with specific primers S, and Si.
Figure3. PCRamplificationofgenomicDNAusingHaliotis-sped&c Lanes: (I) molecular weight standards; (2) ca. 1.45 kb genomic PCR
pnmersS, andS:(30cycles). Lanes:11)molecularweightstandards;(2) product; (3) ca. 1.25 kb genomic PCR product; (4) molecular weight
and(3)0.3MgHaliolisspermDNA;(4)0.75^g TetrahymenaDNA;(5) standards.
0.5 Mg Vibrio DNA; (6) 1 Mgsalmon sperm DNA; (7) no DNA. Other
detailsin Materialsand Methods.
mRNA G
cause the from which the protein sequences
from the environment (Morse, 1990a, b). Purification of were identified was extracted from cilia that had been
cilia in milligram quantities from the cultured larvae has purified and washed by four sequential cycles of differ-
allowed us to analyze the chemosensory receptors and ential centrifugation, it was probably not contaminated
RNA
signal transducers /// vitro (Baxter, 1991; Baxter and by free released from the larvae. Electron micros-
Morse, in prep.), and (as shown here) to isolate mRNAs copy shows no othercell fragments orcellscontaminating
encodingsome oftheseelements and conductanalysesat the purified cilia (Baxter, 1991; Baxter and Morse, in
the cDNA sequence level. We have shown here that cilia prep.). //; situ hybridization will be required, however, to
purified from //. rufescenslarvae contain polyadenylated verifytheintraciliary localization oftheGet mRNA. Three
mRNA, and that this mRNA includes sequences corre- independent lines ofevidence confirm that the source of
sponding to two Ga signal transducing proteins.
The central role of the G proteins as chemosensory
signal transducers was confirmed by /// vitro studies of P
olfactorycilia isolated from frog(Pace etai, 1985). Jones 1 2 3
G
and Reed (1989) recently have identified a unique |f
from the ciliated sensory epithelium ofthe rat olfactory
mucosa; the sequence of this G protein a subunit was
determined by analysis ofits cDNA. G protein also acts
as the primary chemosensory signal transducer in taste
G
receptor cells in the frog; this protein controls the ac-
tivation ofadenyl cylase, with the resultingsequential ac-
tivation ofa protein kinase and membrane depolarization
(Avenet et ai, 1988). G protein transduction ofchemo-
sensory stimulation in catfish controls an inositol tri-
phosphate (protein kinase) cascade (Huque et ai. 1987).
mRNA
Although distal localization of has been ob-
served in other systems (Merlie and Sanes, 1985; Garner
et ai. 1988; Kosik et ai. 1989), the results reported here
mRNA
are the first ofwhich we are aware in which has
been purified, and the corresponding cDNA synthesized,
amplified, cloned, and sequenced from purified cilia. Be-
G cDNAs FROM LARVAL CILIA 325
the G protein sequences characterized could not have labeling with ADP-ribose catalyzed by cholera toxin
come from bacterial contamination: (1) the mRNA se- (Baxter and Morse, in prep.).
lected was poly A+; (2) the sequence ofGal determined In molluscan larvae, thecilia ofthecephalicapical tuft
DNA
corresponds exactly to a sequence in the genomic and of the propodium have been suggested to mediate
(from sperm) ofHaliotis rufescens: and (3) that genomic chemosensory substratum-recognition and the resulting
sequence contains an intron (at the expected position) controlofmetamorphosis(Raven, 1958; FretterandGra-
that would be lacking from bacterial DNA. That these ham, 1962;Bonar, 1978a, b; Chiaand Koss, 1984; Yool,
sequences were from Haliotis mRNA, and not from pos- 1985). Ciliary receptors have been implicated in this pro-
sible contaminants, was further confirmed by the failure cess in other invertebrate larvae as well (e.g., Laverack,
ofthe unique ciliary sequences to direct amplification of 1968; Carthy and Newell, 1968; Chia and Spaulding,
anyhomologoussequencesincontrol samplesofbacterial, 1972; Siebert, 1974; Zimmer and Wollacott, 1977; Eck-
protozoan, or fish DNA, under conditions in which the elbarger, 1978; Reed and Cloney, 1982; Reed, 1987). The
perfectly homologous sequences were detected and am- ciliatedcellsoftheapical tuftsofHaliotislarvaearestellate
plified from Haliotis genomic (sperm) DNA. neurons that appear anatomically to be primary chemo-
The cDNA sequences that we obtained from mRNA sensory receptorcells(Yool, 1985). These neurons project
purified from the cilia isolated from Haliotis rufescens axons to the cephalic ganglia; they also send lateral den-
larvae reveal twoapparent G protein subunit sequences. drites to a pair of adjacent mucus gland cells that are
Although definitive characterization must await comple- stimulated to secrete theircontents in one ofthe first cel-
tion ofthe entire translated sequences, our identification lular changes observed in metamorphosis (Morse et al.,
ofthese sequences as members ofthe G protein family is 1980a; Yool. 1985). These neurons and their cilia dis-
strengthened bythe findingthatGal isvirtuallyidentical appear from the organism after induction of metamor-
(50/51 residues) to Gqa in the region sequenced. and the phosis; the time ofthislosscoincideswith thetimeofloss
observation that the most highly conserved amino acids ofthelabeled morphogenicreceptors(Trapido-Rosenthal
in this region of the other known Ga proteins also are and Morse, 1986a), supporting the suggestion that some
conserved in the Gal and Ga2 sequences from the Hal- of the biochemically labeled chemosensory receptors
iotislarvae (Fig. 2). The genomic sequences that we thus controlling metamorphosis may be located on these cells
far have characterized correspond exactly to theircDNAs, or their cilia.
and contain the intron at the same position between the Ourfindingthatsufficient mRNAcanbeobtainedfrom
G and G' domains as those found in mammalian G pro- the cilia of Haliotis larvae to establish a cDNA library
tein genes. opens this system to analyses, similar to that reported
The two Ga sequences obtained from the cilia ofHal- here, of the cDNAs for the receptors and other signal
iotis larvae are clearly related to the Go sequences from transducersofthe morphogeneticandamplifierpathways
other species (Fig. 2) (and unrelated to tubulin, for ex- that control metamorphosis. These analyses should pro-
ample). Yet the two sequences differ significantly from vide insights into the mechanisms ofaction, functional
one another. The larval cilia Gal sequence is highly ho- integration, andevolutionofthechemoreceptorsandtheir
mologoustothatofthecorrespondingdomainofthealpha associated signal-transducers in the molluscan larvae.
subunit ofthe mammalian Gq. This is of particular in-
terest, in light ofthe finding that Gq is a pertussis toxin- Acknowledgments
insensitive regulator ofphospholipase C (Strathmann and This research was supported by grants from the Na-
Simon, 1990; Smrcka et al, 1991), and our observation tional Science Foundation (DCB-87-18224), theOfficeof
that the lysine-dependent regulatory pathway in Haliotis Naval Research Molecular Biology Program (NOOO14-87-
G
larvae is mediated by a pertussis toxin-insensitive pro- K-0762),andtheUniversityofCaliforniaatSantaBarbara
tein-phospholipase C-protein kinase cascade (Baxter and Training Grant in Marine Biotechnology. We especially
Morse, 1987; Baxter, 1991; Baxter and Morse, in prep.). appreciatetheexpertadvice,andtimeandeffortprovided
Gal is markedly different from G,, Gs, G , Gx, and G ir by Jay C. Groppe. We also gratefully acknowledge the
characterized from othersystems, whereasthe larval cilia helpful suggestions provided by Mel Simon, Thomas T.
Go2 sequence is significantly more closely related to G, Amatruda, and John Sninsky; gifts of genomic DNA
and G (from Drosophila and rat) than it isto the Gsand samples provided by Richard Showalter, Jennifer Ortiz,
G |ffrom thesespecies. Wearenowintheprocessofiden- and Jay Groppe; and expert instruction and assistance in
Ga
tifyingthe sequencecorrespondingtothetransducing the dissection ofretinas, provided by Page Erickson.
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