Proc. Natl. Acad. Sci. USAVol. 84, pp. 5115-5119, August 1987Biochemistry

Human cDNA clones for an a subunit of Gisignal-transduction protein

(receptors/adenylate cyclase/GTP-binding proteins/brain/mRNA)

P. BRAY*, A. CARTERt, V. Guo*, C. PUCKETTP, J. KAMHOLZt, A. SPIEGELt, AND M. NIRENBERG**Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, tMetabolic Diseases Branch, National Institute of Diabetes, Digestive andKidney Diseases, tLaboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20892

Contributed by M. Nirenberg, March 27, 1987

ABSTRACT Two cDNA clones were obtained from a XgtllcDNA human brain library that correspond to a; subunits ofGsignal-transduction proteins (where aj subunits refer to the asubunits of G proteins that inhibit adenylate cyclase). Thenucleotide sequence of human brain a; is highly homologous tothat of bovine brain a; [Nukada, T., Tanabe, T., Takahashi,H., Noda, M., Haga, K., Haga, T., Ichiyama, A., Kangawa,K., Hiranaga, M., Matsuo, H. & Numa, S. (1986) FEBS Lett.197, 305-310] and the predicted amino acid sequences areidentical. However, human and bovine brain a; cDNAs differsignificantly from a; cDNAs from human monocytes, ratglioma, and mouse macrophages in amino acid (88% homol-ogy) and nucleotide (71-75% homology) sequences. In addi-tion, the nucleotide sequences of the 3' untranslated regions ofhuman and bovine brain a; cDNAs differ markedly from thesequences of human monocyte, rat glioma, and mouse macro-phage a; cDNAs. These results suggest there are at least twoclasses of a; mRNA.

Guanine nucleotide-binding proteins (G proteins) couplereceptors for extracellular signals to effectors such as ade-nylate cyclase (1) or cGMP phosphodiesterase (2). G proteinsconsist of three protein subunits, a, /3, and y. a Subunits bindand hydrolyze GTP (1, 2) and display specificity for receptorsand effectors. Different proteins, Gs and Gi, mediate stimu-lation and inhibition, respectively, of adenylate cyclase(where as and a, are the corresponding a subunits). Gs andone or more forms of G1 are assumed to be present in mostmammalian cells (1), whereas the at-1 subunit of transducinis expressed only in retinal rods (3, 4) and at-2 is expressedonly in cones (4). Similarly, ao (a G protein of unknownfunction) is abundant in brain but not in most of the othertissues that have been examined (5, 6).The nucleotide sequences of cDNA clones for bovine (7,

8), rat (ref. 9; R. Reed, personal communication), mouse (10),and human as (11, 12) have been reported. R. Reed andcoworkers have cloned and sequenced three types of aicDNA from a rat olfactory epithelium XgtlO cDNA library(personal communication). Other a, cDNAs from bovinebrain (13), bovine pituitary (14), human monocyte (15),mouse macrophage (10), and rat C6 glioma (9) have beensequenced. In addition, the sequences of rat (9) and bovine(16) ao and bovine at-i (17-19) and at-2 (20) cDNAs have beenreported. The amino acid sequence homologies of a subunitsrange from -40% (as vs. a) to -78% (at-i vs. at-2).

In this report, the nucleotide sequence of a human brain a,cDNA is described and is compared with sequences ofhumanmonocyte (15), bovine brain (13) and pituitary (14), rat C6glioma (9), and mouse macrophage (10) a, cDNAs. Two typesof ac can be distinguished that differ in 12% of the amino acid

--

-* 3*---- -*0--2-

4*A

EM M MMM M M M E.1 200 400 600 800 1000 1200 1400-o 2 4-4 .0-- _ ,_ __* -a 2 *4 14

*4 2 * e---- * * *2

4-2- -t4 -of 2-

FIG. 1. Restriction fragments of BG-4 and BG21-2 ai cDNAswere subcloned into M13mpl8 and sequenced. Each arrow repre-sents a subcloned DNA restriction fragment that was sequenced;arrow shafts composed of dashes represent nucleotide sequencesfrom BG21-2 ai cDNA; those with unbroken shafts representsequences of BG-4 aicDNA. The numbers shown with some arrowsrepresent the number of subclones of the same type that weresequenced. The number of nucleotide residues in human brain a1cDNA is shown on the scale. E and M represent sites cleaved byEcoRI and Mbo I endonucleases, respectively.

residues and possess markedly different 5' and 3' untrans-lated sequences that have been conserved during evolution.

METHODSA Xgtll cDNA library was constructed by a modification ofthe method ofHuynh et al. (21). Poly(A)+ RNA was preparedfrom basal ganglia dissected from a 1-day-old human femalebrain and was used for cDNA synthesis. Duplex DNA >800nucleotide pairs in length was ligated to Xgtll arms that hadbeen dephosphorylated, and the DNA was packaged. Theresulting library contains 106 cDNA recombinants; 90% ofthe phage contain DNA inserts.

Twenty-five thousand phage and 109 Escherichia coliY1090 cells were plated per 150-mm Petri dish. Plates wereincubated at 42°C for 2 hr and then at 38°C for 4 hr. PhageDNA was transferred to replicate nitrocellulose filters thatwere incubated in a solution containing 750mM NaCl/75mMsodium citrate, 1 mg of bovine serum albumin per ml, 1 mgof polyvinylpyrrolidone per ml, 1 mg of Ficoll per ml, 50mMsodium phosphate (pH 6.8), 1 mM sodium pyrophosphate, 50,ug of yeast tRNA per ml, and 20% formamide for 16 hr at42°C. Two probes, designed to hybridize to highly conservedregions of G-a subunit cDNAs (22), were synthesized. Oneprobe, 43 nucleotide residues in length, consisted of 32species of oligodeoxynucleotides, each containing six to eight

Abbreviations: a, and aj, a subunits of guanine nucleotide-bindingproteins (G proteins) that activate (G,) or inhibit (G.) adenylatecyclase; at-1, a subunit of transducin, a G protein of rod photo-receptor cells that activates cGMP phosphodiesterase; at-2, asubunit of transducin, a G protein of cone photoreceptor cells; a0, asubunit of G., a G protein of unknown function.

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

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5116 Biochemistry: Bray et al.

T C Adeoxyinosine residues (5' TCATCTGCTT I ACIATIGTGj-CTTTTCCCIGATTCICCIGCICC 3').The other probe consisted of a single species of oligode-

oxynucleotide 50 nucleotide residues in length (5' ACCT-TGAAGATGATGGCGGTCACGTCCTCGAAGCCGTG-GATCCACTTCTT 3').The 5' terminal hydroxyl groups of the probes were labeled

with 32p from [32P]ATP catalyzed by polynucleotide kinase.Each probe (""1.5 x 106 cpm/ml, 150 fmol/ml) was added tosets of four replicate 137-mm filters and incubated for 16 hr at42°C. Each filter was washed three times in a solution contain-ing 60 mM NaCl/6 mM sodium citrate and 0.1% NaDodSO4 at23°C for 20 min per wash, then washed once at 42°C in 60 mMNaCl/6 mM sodium citrate and 0.1% NaDodSO4 for 3 min, andthen subjected to autoradiography.Phage from plaques yielding positive autoradiographic

signals with both probes were cloned. DNA inserts were

Proc. Natl. Acad. Sci. USA 84 (1987)

excised with EcoRI and subcloned into M13mpl8, and partialnucleotide sequences of the subcloned single-stranded phageDNA inserts were determined by the dideoxynucleotidesequencing method (23). The complete nucleotide sequenceof clone BG-4 was obtained by use of specific syntheticoligonucleotide primers and by sequencing BG-4 DNA frag-ments partially cleaved by Mbo I endonuclease and thensubcloned.Nucleotide or amino acid residues were aligned by using

the NUCALN orPRTALN algorithms of Wilbur and Lipman(24). All amino acid residue alignments were performed witha K-tuple size of 1, window size of 20, and a gap penalty of1. Alignments of nucleotide residues in the coding regionswere performed with a K-tuple size of 3, window size of 20,and a gap penalty of 7; for 3' and 5' untranslated regionalignments a gap penalty of 1 was used.RNA for transfer blots was prepared from adult male

human cerebral cortex and liver (25). Poly(A)+ RNA was

Ser Ala Glu Asp Lys Ala Ala Va1 Clu Arg Sor Lys Met I Asp Arg Ann Lou Arg Cli Asp Cly Clu Lys Ala Ala Arg Clu Val Lys 30AGC GCC GAG CAC AAC aCc CCC CTC 0ac cOc ACT AAC ATC ATC CC CaC AAC CrC COT 01 CAC CC cac AAC acc GccCM C CA CrTC AAC 90

CC C TC AAC C C A C C

Lou Lou Lou Lou Cly Ala Cly Clu Sor Cly Lys Ser Thr no Val Lys CGln lt Lys Ile nl His Clu Ala Cly Tyr Sor Clu Clu Clu 60CTG CTG CTC CTC GGT OCT GOT CGk TCT OCT AAA AT ACA ATT CTM AAC CAj ATC AAA AT? ATC CAT cI CC? OCT TAT TCI CA G01 G010 180T TC C C A C C C C C C c C c A C C C C A

Cys Lys Gln Tyr Lys Ala Val Val Tyr Sar Asn Thr Il.Cle n Sr Ile I1e Ala I1e I1e Arg Ala 11t Cly Arg Lou Lys I1e Asp Pho 90TGT AAA CAA TAC AAA OCT OTC CTC TAC ACT AAC ACC ATC CAC TCA ATT ATT OCT ATC ATT ACM OCT ATO CGG AMC TTC AAC ATA CIC TTT 270C CC C CCC C T C C C C C T CC AL C AACC C C

Gly Asp Sor Ala Arg Ala Asp Asp Ala Arg Cln Lou Phb Val Lou Ala Cly Ala Al Clu Glu Cly Ph* )Mt Thr Ala Clu Lou Ala 119GGT GW TCA CCC CC GCC GCaT CAT cI Cacc CAA CTC Tm CTC CTA OCT aa OCT OCT CAA GCA -- C TTT ATC ACT aCC CAl CT OCT 357

cc C C T AA C C C AC C A CA CTCTCAC CC CCA a C C C C AT C C TCC

Gly Val Il Lys Arg Lou Trp Lys Asp Sor Cly Val Cln Ala Cys Pho Asn Arg Sor Any Clu Tyr CGIn Lou Asn Asp Sor Ala Ala TyrGGC CT? ATA AAC ACX TSC TO ARl CAT ACT OCT CTA CAA CCC TOT TTC AAC Avc TCC CCa GAG TAC CAC CTT AAT CAT TCT GI GC TACC C C CCC OCT CCI C c C T CC CC AAC A C C C A T C

Tyr Lou Asn Asp Lou Asp Arg Il AaCla n Pro Asn Tyr Ib Pro Thr Cln Cl1D Asp Val Lou Arg Thr Arg Val Lys Thr Thr Cly Il1TAT TTC AAT GCA TTC CAC AGl ATA GCT CAA CCA AAT TAC ATC COC AM? CAl CaI GAT CT? CC AGA ACT Aca OTC AAA ACT ACM GCA A=TC C C C OCT T A c ACT GAC C A C C ACO C C C A c C c c C

Val Glu Thr His Pho Thr Ph. Lys Asp Lou His Pho Lys bfst Pho Asp Val Gly Cly CGl Arg Sor Clu Arg Lys Lys Trp 110 His CysGCT GAC ACC CAT TTT ACT TTC AAA CAT CTT CAT TTT All ATC Ti' CIT GM GCC GOCT CAC AGA TCT GAC CCC AAC AA TOO AT? CAT TGCG G A C C C CC A C C c T C C C C

149447

179537

209627

Ph. Glu Gly Val Thr Ala le Il Ph. Cys Val Ala Lou Sor Asp Tyr Asp Lou Val Lou Ala Clu Asp Clu Clu lst Asn Arg Mat His 239TTC GAA IGA GMT ACCACc ATC ATC TTC TOT cTA GCC CTC AM CAC TIC CAC CTC CTT CTA OCT CAA CAT CAA CIA ATC ARC CA ITO CAT 717T C C C A C C C T C C T T C C C C C C

Glu Ser lst Lys Lou Ph. Asp Ser I10 Cys Asn Asn Lys Trp Pho Thr Asp Thr Sor I10 I1 Lou Ph. Lou Asn Lys Lys Asp Lou Pho 269CA ACC ATO AlA TTC TTT GAC ACC ATA T7T ARC AAC AG TOO Ti' AC CAT AMC TCC A=T ATa C? Ti CTA AAC AG MAG CGT CTC TTT 807C C CA C T C C C C C C C C C C C C

Clu Clu Lys 11e Lys Lys Sor Pro Lou Thr 11e Cys Tyr Pro Olu Tyr Ala Cly Sor Ain Thr Tyr Clu Clu Ala Ala Ala Tyr Ib Cln 299CAL CAA AAA ATC AAl AAC AhC CCC CTC AC? ATA TOC TAT CCI CAI TAT GCC CCI TCA AAC ACI TAT CII CA0 WCA OCT GCC TAT AT? CAR 897C C C C CC T C C C TC T C C A C C C A T C ACC C C C

Cys Cln Ph. Clu Asp Lou Asn Lys Arg Lys Asp Thr Lys Clu Il. Tyr Thr His Ph. Thr Cys Ala Thr Asp Thr Lys Asn Val Cli Pho 329TGT CAC Ti' CAA CAC CTC AAT AAA AMl ANO CAC ACM AAC CII ATI TAC AC cac TTC AM TOT CM ACA CGAT AT AAC AR CTC CAC Ti' 987A A C C CC C A C C C C C C C C C C C

Val Ph. Asp Ala Val ¶hr Asp Val Ile no Lys Asn Asn lou Lys Asp Cys Cly lou Phb tezCT? Tm CGA OCT OTA ACA CAT ¢TC A2C ATA IAA ART AlT CTA AAA GCA TOT OCT CTC TT TA CAG C C C C C C C C G C C C C C C CC CC _O OC C

CTATGATACATCATI'?ATC

3491085

1204

1323

1344

FIG. 2. Nucleotide sequence of human brain ai cDNA and predicted amino acid sequence of a, protein. On the third line of each set of linesare shown the nucleotide residues of human monocyte ac cDNA (15) that differ from those of human brain ac cDNA, except for the 3' tail.Nucleotide residues 1-1276 correspond to BG-4 DNA. The regions of BG21-2 DNA that were sequenced correspond to residues 1-500 and959-1344. The underlined nucleotides are the sites of hybridization of the 43-mer and 50-mer 32P-labeled oligodeoxynucleotide probes. The first10 nucleotide residues found in BG-4 DNA are GTGCCGAAAG, whereas the first 11 nucleotide residues found in BG21-2 DNA areTGCCGAAAGCG. We do not know whether these nucleotide residues are cloning artifacts, and therefore these residues are not shown.

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Proc. Natl. Acad. Sci. USA 84 (1987) 5117

L B- Origin

FIG. 3. Transfer blot analysis of poly(A)+RNA (20 ,ug per lane). Lane L, adult human liverRNA; and lane B, adult human brain RNA. The

-3.8 nitrocellulose filter was incubated with a [32P]-* - 2.2 RNA probe corresponding to the minus strand

-1.6 of 3' untranslated region of BG21-2 ai cDNA(nucleotide residues 1062-1344). The dashmarks on the left indicate the chain lengths of

-o 4 the RNA markers used: 9.49, 7.46, 4.40, 2.37,1.35, and 0.33 kilobases; on the right, the chainlengths of the radioactive brain RNA bands areshown.

isolated by oligo(dT)-cellulose column chromatography (26),fractionated by formaldehyde/agarose gel electrophoresis,and then blotted onto BA85 nitrocellulose membranes (Schlei-cher & Schuell). A probe specific for human brain ai mRNAcorresponding to BG-4 or BG21-2 ai cDNA was obtained asfollows: human brain ai cDNA was subcloned into the EcoRIsite of pGEM-blue 3 (Promega Biotec, Madison, WI). Therecombinant replicative form DNA was converted to linearDNA by incubation with Sca I endonuclease; the cleavagesite is 43 nucleotide residues past the termination codon in the3' untranslated region of ai cDNA. The synthesis of a[32P]RNA transcript complementary to 251 nucleotide resi-dues in the 3' untranslated region of human brain ai wascatalyzed by SP6 RNA polymerase (27). The nitrocellulosefilters were prehybridized for 8 hr at 57°C in a solutioncontaining 750 mM NaCl/75 mM sodium citrate, 5 mMsodium phosphate (pH 6.5), 1 mM EDTA, 0.5 mg of bovineserum albumin per ml, 0.5 mg of Ficoll per ml, 0.5 mg ofpolyvinylpyrrolidone per ml, 0.1% NaDodSO4, 200 ,tg ofyeast tRNA per ml, and 50% formamide. The [32P]RNAai-specific probe was added (1 x 106 cpm/ml, 4 fmol/ml) andthe reaction mixture was incubated 18 hr at 57°C. The filterwas washed three times in a solution containing 15 mMNaCl/1.5 mM sodium citrate and 0.1% NaDodSO4 at 55°C,then washed two times at 65°C for 20 min each wash, and thensubjected to autoradiography for 18 hr with an intensifyingscreen.

RESULTS AND DISCUSSIONSequence of Human Brain a, cDNA. A Xgtll cDNA library

prepared from total cellular poly(A)+ RNA from 1-day-oldhuman basal ganglia was screened with two 32P-labeledoligodeoxynucleotide probes complementary to highly con-

H BRAINB BRAINH MONOCYTER C6 GLIOMAM MACROPHAGE

HBBBHMRGMM

HBBBHMRGMM

HBBBHMRGMM

HBBBHMRGNm

served regions of a subunits of G proteins (22). Fourteen ofthe 575,000 cDNA recombinants screened gave positivesignals with both probes. Part of the nucleotide sequence ofeach positive clone was determined, which led to the iden-tification of 2 a, cDNA clones, BG-4 and BG-21-2, and 11 ascDNA clones (11). Both strands of human brain BG-4 a,cDNA and part of BG21-2 cDNA were sequenced (Fig. 1).Most regions of BG-21-2 DNA that were sequenced provedto be identical to the corresponding sequence of BG-4 (withone exception noted in the legend to Fig. 2); however, thechain length of BG21-2 was longer than BG-4. The nucleotidesequence of BG-4 human brain ai cDNA (residues 1-1266)and the additional BG-21-2 sequence (residues 1267-1344) areshown in Fig. 2 and are compared with the recently reportednucleotide sequence of human monocyte a1 cDNA (15). Thefirst nucleotide ofBG-4 corresponds to the 16th residue in thecoding region of human monocyte ai. Nucleotide residues1-1047 comprise an open reading frame coding for 349 aminoacid residues followed by a termination codon and 294additional 3' untranslated nucleotide residues. Two-hundredand seventy-seven of the nucleotide residues scatteredthroughout the coding portion ofhuman brain ai cDNA differfrom those of human monocyte ai cDNA (27%) (15). How-ever, 221 of the nucleotide substitutions are silent mutationsand 56 result in the replacement of 42 of the 349 amino acidresidues compared (12%). Little or no homology was foundin the nucleotide sequences of the 3' untranslated regions ofhuman brain and monocyte ai cDNAs (67% of the residuesdiffer). These results show that the nucleotide sequences ofhuman brain and monocyte ai cDNAs differ substantially andsuggest that human brain and monocyte ai mRNAs are tran-scribed from different genes. These results agree well with thefindings of R. Reed and his colleagues that rat olfactoryepithelium contains three types of ai (personal communication).

Transfer Blot Analysis of Human ai mRNA. A [32P]RNAprobe complementary to nucleotide residues 1062-1344 in the3' untranslated region of BG-21-2 human brain a, cDNA wasincubated with human liver and brain poly(A)+ RNA that hadbeen fractionated by gel electrophoresis and transferred to anitrocellulose filter (Fig. 3). The [32P]RNA probe was ex-pected to hybridize with human brain ai mRNA correspond-ing to BG-4 or BG21-2 cDNA but not to other species ofa-mRNA. Two faint, diffuse bands of radioactive liverpoly(A)+ RNA were detected with chain lengths of 1.7 and1.0 kb, and one major and three minor radioactive bands ofbrain poly(A)+ RNA were found with chain lengths of 2.2,3.8, 1.6, and 0.4 kilobases (kb), respectively. The 3.8-kb a,poly(A)+ RNA from human brain is similar in size to the3.9-kb chain length reported for bovine brain ai mRNA (13).

SAEDKAAVERSKMIDRNLREDGEKAAREVKLLLLGAGESGKSTIVKQMKI IHEAGYSEEECKQYK (70)MGCTL.......................................................... 70....V.......A.......K................................... D.......R--R 70.--0--V........K.....................................D ....R..R 70...V......A.......K............................... D......RR 70

. *

AVVYSNTIQSI IAI IRAMGRLKIDFGDSARADDARQLFVLAGAAEE-GFMTAELAGVIKRLWKDSGVQACFNRSREYQLN (149).......................................... *v*-@@@@v---*-*@---@*@-........................................................ 149..MVK.'/I(* N-Q-..A-PS.........A-SCT--* Q-VLPDD-S--R...e ANH.....G.... . 150.M...M- VY N.Q-. A-PQ........A-SC@..Q*MLPEDS ... R. -H.A...G.... . 150

...........L--VKR N- ...A-PQ.........A-SC **Q. MLPED-S-R ..A* H.....G..... 150* * I

.E.A~~~~~~~~-

.E*.5.A*..

..........E*D.A..............................

(229)229230230230

YDLVLAEDEEMNRMHESMKLFDSICNNKWFTDTSI ILFLNKKDLFEEKIKKSPLTICYPEYAGSNTYEEAAAYIQCQFED (309)................................................................................................................................ 309............................T.....................*TAK.........T-- . S*SK... 310

............................ ......................T ......F.. @.@@**-**TQ-*--TA-K-D-SSK.......S..............SK........... 310

.............. .............. -.---TQ-S-*.F.TA-K-D .. S... .SK-... 310.TS* TAKD..*~K*LNKRKDTKEIYTHFTCATDTKNVQFVFDAVTDVIIKNNLKDCGLF (354)............................................. ......354............................................. ...355............................................. ...355............................................. ......355

FIG. 4. Amino acid sequence of a, fromhuman brain compared with a, sequences frombovine brain (13), human monocytes (15), rat C6glioma cells (9), and mouse macrophages (10).The letters represent the single-letter abbrevia-tions for amino acids. The symbol * representsan amino acid residue that is identical to theresidue shown for human brain aj; - representsa gap.

Biochemistry: Bray et al.

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Proc. Natl. Acad. Sci. USA 84 (1987)

BOVINEHUMANHOUSE

TGGCCGGCGTCAGAGAATTCGAACGCCTGCCGGCAGTCCCGAGTGCTTCCCGCAGAGGGCTG--GTGGTG

B CATCCAGAAAGAAAGAATTCACCTGTGTTTCGAGGCAGCGCGCCGH GGAGCGGAGTGGAGTCGGGCGGGGCCGAAGCCGGGCCGTGGGC-GR C.M.-.G---.C-

B GACTTCGAGGGAGCGGCAGCCAGCTTTCGCTCCTGGCACA ATCH TAQATGGGGGCCGGGCGGCGGCGGAGCGGCGGAACGCGGG ATGR *Go-.99*-A..----- **9GC-.**CG**A** ATGM OG--00*0.00A.-----...GC**-.-CGe*A. ATG

FIG. 5. Nucleotide residues in the 5' untranslated regions ofbovine brain aj-1 cDNA (13) and human monocyte aj-2 cDNA (15).The symbol * represents a nucleotide residue in rat (9) or mouse (10)ai cDNA that is identical to the residue shown in human monocyteai cDNA. Rat and mouse nucleotide residues that differ from thoseof human macrophage cDNA are shown. ATG at the 3' end of eachsequence represents the initiation codon for methionyl-tRNA.

These results reveal tissue-specific differences in the expres-sion of human ai-1 mRNA.Comparison of a; Amino Acid Sequences. The predicted

amino acid sequence of human brain ai is compared in Fig.4 with ai amino acid sequences predicted from nucleotidesequences of cDNAs from bovine brain (13), human mono-cytes (15), rat C6 glioma cells (9), and mouse macrophages(10). The predicted amino acid sequence of human brain ai isidentical to that of bovine brain ai (13) and differs in only 3amino acid residues from bovine pituitary ai (14) (not shown).In contrast, human and bovine brain ai differ from humanmonocyte, rat C6 glioma cells, and mouse ai in -12% of theamino acid residues. The amino acid sequences of a, fromhuman monocytes, rat, and mouse are closely related (99%homology) and contain a codon for an additional amino acidresidue, Gln-117, which is not present in human brain, bovinebrain, or bovine pituitary a, cDNAs. These data reveal twotypes of ai cDNA: aj-1 from human brain, bovine brain (13),and bovine pituitary (14), and as-2 from human monocytes(15), rat C6 glioma cells (9), and mouse macrophages (10).Thirty-six of the 44 amino acid residues of a,-1 and aj-2 thatdiffer are clustered in two regions: region A (amino acidresidues 82-142) with 25 residues that differ and region B(amino acid residues 280-309) with 11 residues that differ.Furthermore, only 55% of the amino acid replacements areconservative replacements (28). Regions A and B contain thegreatest diversity in amino acid sequence in the a family ofproteins and may contain sites that determine the specificityof G-protein interactions with effectors and receptors, re-spectively (29).The predicted secondary structures of a,-1 and a,-2 based

on the parameters of Chou and Fasman (30) differ in inter-esting ways. Amino acid residues 118-124 of a,-1 (thenumbering system is that of bovine brain aj-1 shown in Fig.3) are predicted to form an a-helix that is not present in aj-2.Conversely, amino acid residues 97-100 and 120-123 of

H BRAIN TAAGTTTT-GCAGTCC--ATGGTAAAATGCATTTTCAAACCARATGAGTACTTATATATGGATCTCTGTAB BRAIN TGA-.G.QG*G*-- Aoo ooo-oQ.-@@@*oooooo.o....CoGo--o-o....Q. C..H MONOCYTE TGAGGGGCAGCGGGGCCTGGCGGGATGGGCCACCGCCGAATTTGTACCCCCCACCCCTGAGGAAGATGGR C6 GLIOMA TGA.o--o-Too..-......A.o.....@oToC-C. -CToo. AoT--.TG*o-@3.M MACROPAGCE TGA .. T..-T-A-. A. * TT.CGG.G-CT.TGCC-ACCCAoT.ToTG -GTC.GAGGCCCCA

SB GACTAGAGTCTTGCAGCAACACAGAATGTAATATAGGC TGCATCTGGGACTTGACCAAAGTTGTTCTGTTTTGTTBB *.....Co-o....o-00......... G-.-oT-@CGG@@oso.. -C...o--ooo-A-.....SM GGGCAAGAAGATCACGCTCCCCGCCTGTTCCCCC-GCCGCTTTTCTCCTCTTTCCTCTCTTTGTTCTQGCTCCCCCTGRG.o-oo ..C..-T...oo-T.o-ooo@@@.@A@T-oo-C--C-o-GoC-T-oo--C .o.o-.- o----MM AA.* *AoGC*CA-GAAG GTGAGAGAoA.G ATo AGACAAAGC-ACCTGCTATeC*CG.AG TTTAAAGAAA

SB TTTT---TAACTGAAAGTAACAGAAGACCTTTCTTAAATGTGACAGATGGTCCTGCAGT-TGAACTGAAGGACAGTGBB .-.o.. o-.oo@o.... TG .--.-o.o-.-oG-.*G.-@@G-oGe-...-.-.-G..........G--.--SM TCCCCTCA----GCTCCAAACGTAGG-GGAGGGGTTCGCACAGGCCTCCCTGTTTGAAGCCTGCCCTTGTCTGAGAT-GRG .--....CCTCG *o-TG.-T-T.-.-.G......GC..............CoA.-A-ACA--T..-oo-oG-MM AAAAAGAnAAA

SB TTAAAGCTGGGCTCTAGTATATTQ&TGATTTCTGCATAAGTGTAAATATGCAAATGTATGTATACATGTATTTATGBB . G.*.-oG-oCG.o ..-Co*o-A. CA.--o.o* oo ..................SM CTGGTAATGGCCATGGTACCCCCTT-CTGGGCATCTGTTCTGGTTTT-TAACCATTGTCTTGTTCTGTGATGAGGGRG .C-o.G.G... .... .T...oA- o-- G..C..0-Co--*-*oo eGoG.- -Co

human monocyte, rat, and mouse a1-2 are predicted to formp-turns that are not present in human and bovine a,-1 proteinsubunits. The differences in predicted secondary structuresare located in a variable region of a proteins that is thoughtto interact with effector molecules.The amino acid sequence of human aj-1 is identical to that

of bovine aj-1, whereas the amino acid sequences of human,rat, and mouse as-2 differ from one another by 3-8 amino acidresidues. These amino acid replacements may have resultedfrom relatively recent mutations during the last 8.5 x 107years because human, bovine, and rodent precursors di-verged from a common ancestor -8.5 x 107 years ago (33).However, the amino acid sequences of human and bovineaj-1 differ from the sequences of human, rat, and mouse a1-2in 36 additional amino acid residues, and the mutations thatresulted in these amino acid substitutions must have occurred>8.5 x 107 years ago. Such considerations lead us tospeculate that aj-1 and a1-2 mRNA are transcribed fromseparate genes that originated by duplication of an ancestrala1 gene much more than 8.5 x 107 years ago and then divergedover a long period of time by accumulation of mutations. Thedifferences between the amino acid sequences of as-1 and aj-2are likely to be functionally significant, since the differencesapparently have been conserved during evolution. In someways the relatedness of aj-1 and aj-2 resembles that of at-1(17-19) and at-2 (4, 20), which exhibit 78% amino acidhomology and interact with rhodopsin in retinal rods andopsin pigments in cones, respectively.

a; Nucleotide Sequences. Comparison of a, cDNA nucleotidesequences from different organisms (not shown here) providesadditional evidence for two types of aj. The nucleotide se-quence of human brain a, cDNA closely resembles that ofbovine brain (13) and bovine pituitary (14) a, cDNAs (94%homology); in addition, human monocyte (15), rat C6 glioma (9),and mouse macrophage (10) aj-2 cDNA nucleotide sequencesclosely resemble one another (87-90% homology). However,aj-1 nucleotide sequences differ substantially from those of a,-2.As shown in Fig. 5, the nucleotide sequence of the 5'

untranslated region of bovine brain as-1 differs markedly fromthe corresponding sequences of human monocyte, rat C6glioma, and mouse macrophage aj-2 cDNAs (33% homology).However, the 5' untranslated nucleotide sequences of a,-2cDNAs from human monocytes, rat, and mouse closely resem-ble one another, which suggests that the a,-2 5' untranslatednucleotide sequences have been conserved during evolution.Comparison of the initial nucleotide residues in the 3'

untranslated regions of ai cDNAs (Fig. 6) shows that humanand bovine brain (13) ai cDNAs are closely related (92%homology) and that human monocyte and rat ai cDNAs arerelated to one another (83% homology). However, little or nohomology was detected between the 3' untranslated regionsof human brain and bovine brain ai cDNAs compared to

FIG. 6. Nucleotide sequences at the beginning ofthe 3' untranslated regions of human brain at-1 andhuman monocyte a,-2 cDNAs (15). The nucleotidesequence of bovine brain aj-1 cDNA (13) is comparedwith the sequence of human brain at-1 cDNA, whereasthe nucleotide sequences of rat (9) and mouse (10) aj-2cDNAs are compared to the nucleotide sequence ofhuman monocyte a,-2 cDNA. TAA or TGA at the 5'terminus represents termination codons for ai cDNAs.The symbol * represents a nucleotide residue that isidentical to the residue shown for human brain a,-1 orhuman monocyte ac-2 cDNA. Nucleotide residues thatdiffer are shown.

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Proc. Natl. Acad. Sci. USA 84 (1987) 5119

HUMAN TAAGAAGGGAACCCCCAAATTTAATTAAAGCCTTAACCACBOVINE TAA-eeP-e-.o*T.*OG.vvv@C .C.*o-e*GRAT TAA . C.C*.**G@@---@@@@@@*C-e........eMOUSE TAP .A .C-*****.*C_@@@@--@....H AATTAATTAAAAGTGAAACGTAATTGTACAAGCAGTTAATCACCB .00000*---00-A-G.TA .--M------C.*-.*G-e-@-R . C. C. *.-.M1. G. G.

H CACCATACCGCATGATTAACAAAGCAACCTTTCCCTT-CcCB.R ..*..0000000000C..@CC..--@@@--@-@@TT.TM .*e.e---oo**C.o..CC .@@**-@@-*T'.TT..

FIG. 7. Nucleotide sequences at the beginning of the 3' untrans-lated regions of a, cDNAs from human brain (11) and human liver(12). The human nucleotide sequence is compared with the se-quences of a, cDNAs from bovine brain (7) and bovine adrenalmedulla (8), which are identical in this region, and with rat C6 glioma(9) and mouse macrophage (10) cDNAs. TAA at the 5' terminusrepresents the termination codon for a. cDNA. The symbol vrepresents a nucleotide residue that is identical to that shown forhuman a, cDNA; nucleotide residues that differ are shown.

human monocyte, rat, and mouse ai-2 cDNAs. The sequenceof the first 25 nucleotide residues from the 3' untranslatedregion of mouse as cDNA matches the initial 3' untranslatedsequences of human monocyte and rat ai cDNAs (92-96%homology), but thereafter, the sequences are not related.As shown in Fig. 7, the nucleotide sequences of the 3'

untranslated regions of human, rat, mouse, and bovine ascDNAs also are highly conserved (90-93% homology). How-ever, the initial portion of the 3' untranslated nucleotidesequence of human brain as cDNA (11, 12) is not related tothe 3' untranslated sequences ofhuman ai-1 or ai-2, rat ao (9),or bovine at-1 (17-19) or at-2 (20) cDNAs (not shown). Therelatively high homologies in untranslated regions of ai-1,ai-2, or a, mRNAs in different species suggest that theuntranslated nucleotide sequences are functional and thushave been conserved during evolution.The 3'-terminal untranslated region of bovine brain ai-1

cDNA contains many repeats of (A+T)-rich sequencessimilar to the consensus sequences TTATTTAT (34) andTT(G/A)NNNTTTTTTT (35), which have been found in the3' untranslated regions of some species of mRNA and havebeen proposed to function as signals for rapid turnover ofmRNA (36). The (A+T)-rich sequences are less frequent inao and a, and have not been found in human, rat, or mouseai-2 cDNAs. Whether ai-1 mRNA turns over more rapidlythan ai-2 mRNA remains to be determined.The nucleotide sequences in the 3' untranslated regions of

,3-actin, cardiac a-actin, c-fos, nerve growth factor, andcreatine kinase mRNAs from different organisms also havebeen conserved during evolution (see ref. 37 for discussion).Different forms of actin and creatine kinase with conserved3' untranslated regions have been shown to be the productsof separate genes that are expressed in different tissues andat different times during development.Data from a cDNAs have revealed an unexpected diversity

in ai and a, (7-18). Comparison of human brain and humanmonocyte (15) as cDNAs suggests that the two types of humanas are transcribed from separate genes. The nucleotide se-quences of ai cDNAs reveal that ai genes are subject to strongselective pressure in the coding region and the 5' and 3'untranslated regions. Comparison of amino acid sequencespredicted from a, cDNAs suggests that as-1 and ai-2 proteinsmay differ in function as well as in tissue distribution andabundance.

We thank Dr. Gerald Zon for synthesizing the oligodeoxynucleo-tide probes. This work has been presented by P.B. in partial fulfillmentof the Ph.D. requirements of George Washington University (Wash-ington, DC).

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