Proc. Nati Acad. Sci. USAVol. 79, pp. 5901-5905, October 1982Cell Biology

Molecular structure and evolutionary origin of human cardiacmuscle actin gene

(recombinant DNA/DNA sequence/intron location/gene family/stretched poly[d(T-G)] sequence)

HIROSHI HAMADA, MARIANNE G. PETRINO, AND TAKEO KAKUNAGACell Genetics Section, Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205

Communicated by Peter K. Vogt, June 10, 1982

ABSTRACT Two recombinant phages that contain cardiacmuscle actin gene were isolated from a human DNA library andtheir structures were determined. Restriction analysis indicatesthat both clones carry the sameEcoRI 13-kilobase fragment wherethe coding sequence is mapped. The cloned DNA hybridized withpolyadenylylated RNA from human fibroblasts, which directs thesynthesis of cytoplasmic (3- and y-actin in vitro. However, se-quence determination of the cloned DNA showed that the entirecoding sequence perfectly matched the amino acid sequence ofcardiac muscle actin. The initiation codon is followed by a cysteinecodon that is not found at the amino-terminal site of any actin iso-form, suggesting the necessity of post-translational processing forin vivo actin synthesis. There are five introns interrupting exonsat codons 41/42, 150, 204, 267, and 327/328. Surprisingly, theseintron locations are exactly the same as those of the rat skeletalmuscle actin gene but different from those of nonmuscle g3-actingene. Nucleotide sequences of all exon/intron boundaries agreewith the G-T/A-G rule (G-T at the 5' and A-G at the 3' terminiof each intron). The 3'-untranslated sequence has no homology tothat of nonmuscle P- or y-actin gene, but Southern blot hybrid-ization has shown that this region has considerable homology tothat ofone of the other actin genes. These results indicate that therecombinant phages, which we have isolated, contain cardiac mus-cle actin gene and that cardiac muscle actin gene and skeletalmuscle actin genes are derived from their ancestor gene at a rel-atively recent time in evolutionary development.

Six actin isoforms are known in vertebrates-four muscle types(skeletal, cardiac, and two smooth muscle types) and two non-muscle types (1). Each muscle-type actin is functionally in-volved in muscle contraction and is expressed only in particularmuscle tissues. On the other hand, nonmuscle actins, so-calledcytoplasmic actins, participate in a variety of functions such ascell motility, mitosis, and maintenance of the cytoskeleton andare expressed in all kinds of cells (2, 3). Abnormal expressionof cytoplasmic actin has been found to be associated with neo-plastic transformation of human fibroblasts (4-7).

It was expected from protein analysis that vertebrates haveat least six actin genes (1). However, little is known about thearrangement of actin genes in the vertebrate genome, althoughthe existence of multiple actin genes has been shown in lowereukaryotes such as Dictyostelium, Drosophila, and sea urchinby directly isolating the genes (8, 9). Thus, a study of the struc-ture and organization of the human actin genes is necessary tounderstand the regulation of their differential expression, theevolutionary origin of these related genes, and the possible in-volvement of their abnormal expression in diseases.

This paper describes the isolation and characterization of ahuman cardiac muscle actin gene. The structural features andthe evolutionary origin of this gene are discussed.

MATERIALS AND METHODS

Materials and Cells. pcDd actin ITL-1 (pcDd), a recombi-nant plasmid containing a cDNA copy of actin mRNA fromDictyostelium discoideum, was obtained from R. A. Firtel(Univ. of California). HeLa cells were obtained from the Amer-ican Type Culture Collection (Rockville, MD). HuT-14 cells arean in vitro chemically transformed human fibroblast cell line(5, 10).

Screening of DNA Library. The genomic library (con-structed by partial EcoRI digestion of DNA from a 13-thalasse-mia patient and cloned into the Charon-4A phage vector) wasthat of Fritsch et aL (11). This library was screened as describedby Benton and Davis (12) with the Sau96 I 0.85-kilobase (kb)fragment of pcDd DNA as probe (13); this probe was radioac-tively labeled by nick-translation (14). Plaque hybridization wascarried out in 6x NaCI/Cit (lx NaCl/Cit = 0.15 M NaCl/0.015 M Na citrate, pH 7) containing 0.08% polyvinylpyrroli-done, 0.08% Ficoll, and 0.8% bovine serum albumin at 65°C.Filters were washed first in 2X NaCl/Cit containing 0.1%NaDodSO4 at 50°C three times and then either in 0.1 x NaCl/Cit containing 0.1% NaDodSO4 at 50°C ("stringent condition")or in 0.2x NaCl/Cit containing 0.1% NaDodSO4 at 37°C ("lessstringent condition") twice. All the phages that showed positivesignals in the first autoradiograms were picked up and screenedone more time. Two phages that showed positive signals in thesecond screening were purified by repeated plating and plaquehybridization until 100% of the phage were positive. PhageDNA was prepared as described by Lawn et al. (15). All exper-iments involving recombinant phages and plasmids were car-ried out in accordance with National Institutes of Health re-combinant DNA guidelines.

Restriction Mapping. Restriction enzymes were purchasedfrom Bethesda Research Laboratories and utilized according totheir recommended assay procedures. Digested phage DNAswere electrophoresed on a 1% agarose gel and transferred toa nitrocellulose filter by the method of Southern (16). Filterswere hybridized to a 32P-labeled probe as described above. Thefinal washing was carried out under the less stringent condi-tions. For the detailed mapping of smaller fragments subclonedin pBR322, the method of Smith and Birnstiel (17) was used.

Subcloning Strategy. Smaller fragments ofinterest were sub-cloned in pBR322. All subcloned fragments originated fromphage AHa-25 DNA: the EcoRI-BamilI 8.0-kb DNA fragmentwas inserted between the EcoRI and the BamHI sites ofpBR322. The Pst I 2.2-, 0.9-, 0.4-, 0.9-, and 0.6-kb DNA frag-ments (see Fig. 2)-were inserted at the Pst I site of pBR322.DNA Sequence Determination. The chemical base-modifi-

cation/cleavage procedure ofMaxam and Gilbert (18) was used.Five reactions were done for each end-labeled sample (G, G+A

Abbreviations: kb, kilobase; pcDd, recombinant plasmid pcDd actinITL-1; NaCl/Cit, 0.15 M NaCl/0.015 M Na citrate, pH 7.

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5902 Cell Biology: Hamada et aL

A+C, T+C, and C). The autoradiograms were read indepen-dently by two people.

RESULTSIsolation and Restriction Mapping of Clones. A human DNA

library constructed from partial EcoRI-digested DNA (11) wasscreened with a Dictyostelium actin cDNA probe (13) labeledwith 32P by nick-translation. When the filters were washed un-der the stringent hybridization conditions, no clones that hy-bridized to this probe were detected among _106 phagesscreened. However, when the filters were washed under theless stringent conditions, two clones (designated AHa-25 andAHa-38) were obtained.DNA was prepared from each clone, digested with various

restriction enzymes, analyzed on an agarose gel, and hybridizedto pcDd [32P]DNA. Some DNA fragments hybridized with theprobe under the less stringent condition (Fig. 1). When the fil-ter was washed under stringent conditions, the intensity of thehybridized bands greatly decreased (data not shown).

Thus, restriction maps were made for both AHa-25 and AHa-38 (Fig. 2A). Both clones had the overlapping hybridizableEcoRI 13-kb fragment. Their 13-kb region made perfect du-plexes in heteroduplex analysis (data not shown). It is likely,therefore, that the human cell DNAs in AHa-25 and AHa-38were derived from the same chromosomal locus. Thus, AHa-25was chosen for further studies, unless otherwise specified.

Hybridization with Human Actin mRNA. The EcoRd-BamHI8-kb fragment of AHa-25, which strongly hybridized with thepcDd probe, was subcloned in pBR322 DNA. This subelonedDNA, designated pEB8.0, was hybridized to polyadenylylatedRNA from the human transformed fibroblast cell line HuT-14.Hybridized RNAs were then characterized by analyzing the invitro translation products directed by them (Fig. 3). Polyaden-ylylated RNA directed many proteins, including the actins (Fig.3A, lane T; Fig. 3B). On the other hand, the hybridized RNAdirected the synthesis of only actins (Fig. 3A, lane H; Fig. 3C).We have identified the polypeptides that migrate at the posi-

B H

M E B E H E

23.7

9.7

6.6

4.3

2.2

0.5

kbA

B H

E B E H E

B

FIG. 1. Restriction digestions and blot hybridizations. (A) AHa-25DNA was digested with single or double restriction enzymes, electro-phoresed in 0.7% agarose gel, and stained with ethidium bromide. (B)After transfer to nitrocellulose filter, the DNA was hybridized withpcDd [32P]DNA, and the filter was washed under the less stringentcondition. Restriction enzymes were EcoRI (E), BamHI (B), andHindiI (H). Hindll digest of ADNA served as a molecular standard(M). An approximate size scale is indicated on the left.

AHae-5

AHa-38 R

5 10 15 2D 25i i, (Kb)

--

------ L

9? T ? I T? ?

R

A A d 6 AA

99 99 9 9-e? v v v q v v 9f

lKb0-.6 V 5 IV 41I3 11 2

0. .-1 0. I .-2

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1

2.2

AH AAH HH H HHH HV, I ( I I I 11 II

H

III AX SHp S Hp I'HpS S SHp

A

B

C

| - 4 1Kb

FIG. 2. Restriction maps and strategy for sequence determination.(A) Restriction maps of AHa-25 and AHa-38. The map was constructedfrom the blot hybridization analysis shown in Fig. 1, showing thecloned human DNA segment ( ), the Charon 4A arms (R = rightand L = left) (---), and the region hybridizable to pcDd DNA probe(n). Restriction enzyme cleavage sites: 9,EcoRI; I, BamHI; 4, Hindg;4, Sma I; 9, Xba I. (B) Detailed restriction map of the EcoRl-BamHI8-kb fragment pEB8.0. The Pst I site (Y) was determined as describedin A. m, Coding regions whose locations were confirmed by DNA se-quencing. The arrow indicates the direction of transcription. Six exonsand five introns are designated 1-6, and I-V, respectively, from the5' end to the 3' end. (C) Strategy for sequence determination. Each PstI fragment inB (2.2,0.9, 0.4, 0.9, and 0.6 kb) was subcloned in pBR322,and used for sequence analysis. Arrows indicate the direction and ex-tent of sequence determination of each fragment analyzed. The fol-lowing restriction enzyme cleavage sites were used to label the 5'-endsof the DNA fragments: H, Hinfl; A, Alu I; S, Sau96 I; Hp, Hpa II; X,Xba I. Alu I sites in the Pst I 2.2-kb fragment were not determined.The solid bars under the restriction map indicate the regions whosesequences are determined here and shown in Fig. 4. The wavy linedenotes the Hinfl 0.3-kb fragment that was used as hybridizationprobe in Fig. SB.

tions indicated on the gel as f3, y-, and Ax-actins (5). AX-actinis a mutated f3-actin with a one amino acid substitution ex-pressed in HuT-14 cells (5, 7). These results indicated that theDNA sequence of AHa-25 had homology with human /3- and y-actin mRNA.

Nucleotide Sequence of the Coding Region. The amino acidsequences of actin proteins (1) have suggested that vertebrateshave at least six actin genes. A recent report (19) suggests theexistence of 20-30 copies ofactin or actin-like genes. Therefore,we next determined the DNA sequence, especially of the cod-ing region, to identify the actin isoform that the cloned geneencoded. The primary nucleotide sequence ofthe entire codingregion and of some of the introns was determined by the strat-egy shown in Fig. 2C. The differences in the amino acid se-quences are small among the six actin isoforms whose entireamino acid sequences are known (1). The DNA sequence oftheentire coding region of AHa-25 perfectly matched the aminoacid sequence of cardiac muscle actin but not of the other iso-forms (Fig. 4). The amino-terminal polypeptide sequence var-ied the most between the different actin isoforms. The nucleo-tide sequence after the initiation codon agreed with the amino-

-JEL------MELJ- m -Mmma.. W

Proc. Nad Acad. Sci. USA 79 (1982)

Proc. NatL Acad. Sci. USA 79 (1982) 5903

Actin

A C

FIG. 3. In vitro translation of AHa-25-selected RNA. pEB8.0 DNA(100 jig; shown in Fig. 2B) was hybridized to 50,ug ofpolyadenylylatedRNA from a human transformed fibroblast cell line, HuT-14 (10), andhybridized RNA was translated in vitro by reticulocyte lysate as de-scribed (5). (A) NaDodSO4/polyacrylamide gel electrophoresis of invitro products directed by endogenous RNA in reticulocyte lysate (lane0), polyadenylylated RNA (lane T), and pEB8.0-selected RNA (laneH). (B andC) Two-dimensional gel patterns of in vitroproducts of lanesT and H, respectively; 8, e, and ax, the unacetylated precursor for .,

fy, and Ax, respectively; E, the product directed by endogenous RNA.

terminal polypeptide sequence of cardiac muscle actin exceptthat one cysteine codon was found after the initiation codon.

All vertebrate actins thus far characterized begin with an as-

partic or glutamic acid that is acetylated, and the cysteine hasnot been found at the amino terminus of any known actin iso-form (1). On the other hand, a cysteine codon has been foundat this position in all six Drosophila actin genes (9) but not inthe Dictyostelium or yeast actin genes (8). Thus, the presenceof a single extra amino acid residue may be a common charac-teristic of vertebrate actin genes, suggesting the necessity ofpost-translational processing for in vivo actin synthesis.

Location and Sequence of Introns. DNA sequence deter-mination showed the presence of five introns (I, =1.0 kb; II,300 bp; III, 150 bp; IV, 0.6 kb; and V, -0.5 kb) that interruptthe coding region at codons 41/42, 150, 204, 267, and 327/328, respectively (Figs. 2 and 4). The border sequences of allfive introns are in good agreement with the consensus sequencefor such regions (20). Each intron begins with G-T at the 5' ter-minus and ends with A-G at the 3' terminus. A simple repetitivesequence, a (dT-dG)25, was found next to the 3'-splicing site ofintron IV, and also a shorter repeat of the dinucleotide was lo-cated close to the 5' splicing site of intron III.

Nucleotide Sequence of the 3'-Untranslated Region. Thesequence A-A-T-A-A-A, characteristically found in most eukar-yotic polyadenylylated mRNAs -20 bases upstream from thepolyadenylylation site, was found 151 bases downstream fromthe termination codon. At 39 bases downstream from A-A-T-A-A-A, there was a stretch of five Ts, which have been observedin the 3' flanking regions ofsome, but not all, eukaryotic genes.Thus, the 3' end of this gene is likely to be located between theA-A-T-A-A-A and the 5-T stretches. The total length of the 3'untranslated sequence, is thus 160-200 bases (although theprimary structure ofcardiac muscle actin mRNA is not known).cDNA from (-or 'y-actinmRNA hybridized to the coding regionof the cardiac muscle actin gene but not to its 3' untranslatedregion under the less stringent hybridization condition (data not

shown). Therefore, the 3' untranslated region is not homolo-gous between the cardiac muscle actin gene and the cytoplasmicactin genes.

Southern Blot Analysis of Cardiac Muscle Actin Genes. Theorganization of the cardiac muscle actin gene in the human ge-nome and its sequence homology to other actin genes were ex-amined by Southern blot analysis with three different [32P]DNAfragments of cloned DNA as hybridization probes. When thePst I 2.2-kb fragment shown in Fig. 2B containing the codingsequence (codons 1-150) was used as a hybridization probe,more than 20 bands, including the 13-kb fragment derived fromthe cardiac muscle actin gene, were observed in the EcoRI-di-gested human DNA (Fig. 5A). On the other hand, the Pst I 0.4-kb fragment located in intron IV produced a single band in eachrestricted human DNA: 13 kb in the EcoRI digest, 2.1 kb in theXba I digest, and 0.7 kb in the HinfI digest (Fig. 5B). In eachdigest, chromosomal DNA and cloned DNA gave the hybridiz-able bands with the same molecular size. The third probe, aHinfl 0.3-kb fragment of the 3' untranslated region of AHa-25,as shown in Fig. 2C, detected two bands in the Xba I-digestedhuman DNA (i.e., the 2.1-kb band, which represented cardiacmuscle actin gene, and the 1.5-kb band).

DISCUSSIONThe results indicate that two overlapping recombinant phagescontain the human cardiac muscle actin gene. The comparisonofthe intron location in the cardiac muscle actin gene with thosein other actin genes suggests how these genes may haveevolved. Chicken skeletal muscle actin genes have been shownto have five introns at codons 41/42, 150, 204, 267, and 327/328 (21). Rat skeletal actin gene has the same intron pattern(22). These locations are exactly the same as those ofthe humancardiac muscle actin gene, indicating that skeletal and cardiacmuscle genes may have arisen from a single ancestor by geneduplication at a relatively recent time during evolution.

It is probable that all muscle actin genes (i.e., skeletal, car-diac, and smooth muscle actin genes) have come from a singleancestral muscle actin gene. Partial DNA sequence determi-nation ofthe human smooth muscle actin gene has indicated thatit has intron locations similar to those ofthe skeletal and cardiacmuscle actin genes (at least at codon 267 and 327/328; unpub-lished data). It has been reported that all members of the vi-tellogenin gene family (23) and ovalbumin X-Y gene family (24)have the same intron locations. Furthermore, rat cytoplasmicP-actin gene is reported to have a different intron location (i.e.,at least two introns at codons 120 and 267 (22). Because Dro-sophila and Dictyostelium actin genes more closely resemblethe cytoplasmic actin gene than the skeletal and cardiac muscleactin genes in terms of nucleotide sequence homology of theircoding regions (8, 9), it is very likely that the cytoplasmic-typeactin genes arose long before the appearance ofvertebrate mus-cle-type actin genes. In contrast, the distinct intron pattern ofthe muscle-type actin genes indicates that, subsequent to thedivergence of the ancestral muscle and cytoplasmic-type actingenes, the muscle-type actin genes underwent considerablealteration in gene structure. On the other hand, the presenceofthe intron at codon 267 is common to all ofthe vertebrate actingenes whose DNA sequences have been analyzed-i.e., humancardiac muscle (this report), rat skeletal muscle and cytoplasmic(- (22), and chicken skeletal (21) actin genes. This interruptionmay reflect the functional domains of the actin protein as sug-gested with other genes (25, 26).

Southern blotting studies (Fig. 5) led to the following fourobservations.

(i) The probe containing the 5' coding sequence ofthe cardiac

Cell Biology: Hamada et aL

5904 Cell Biology: Hamada et al

hMetCysAspAspGl uGl uThrThrAl &LeuValCysAspAsnCTGCAGAAACCCCCTGAAGCTGTGCCAAGATGTGTGACGACGAGGAGACCACCGCCCTGGTGTGCGACAAC

GlySerGlyLeuVal LysAl aGlyPheAl aGlyAspAspAl aProArgAl aVal PheProSerIleValGlyGGCTETGGGCTGGTGAArGCCr;GrTTTGCGGGCGATGACGCGCCCCGCGCTGTCTTCCCGTCCATCGTGGGC

Exon 1

ArgProArgHi sGl nCGCCCGCGGCACCAGGTAAACTTCCCGCCGAGCCCCCCGTCCCACTCGGGACCCCTTCAGTCCAGCGATCT

AGGAAATGGCTCTCACCT ------------ Intron I

GGTATTTAAATATGTTCCTTGACTTGGGCAGTTAGATATAAATGGACAAGACACTGATTATATTCCTGACA

GlyValMetValGlyMetGlyGl nLysAspSerTGGTGAGAGCATGATTTTCTCATTTTTTCTTCTCATAGGGAGTTATGGTGGGTATGGGTCAGAAGGACTCC

TyrVal GlyAspGl uAl aGlonSerLysArgGlyll eLeuThrLeuLys^TyrProIl eGl uHi sGlyIleIl eTACGTAGGTGATGAAGCCCAGAGCAAGAGAGGCATCCTGACCCTGAAGTATCCCATCGAGCATGGTATCATC

ThrAsnTrpAspAspMetGl uLysIl eTrpHi sHi sThrPheTyrAsnGl uLeuArgValAl aProGl uGl uACCAACTGGGACGACATGGAGAAGATCTGGCACCACACCTTCTACAATGAGCTCCGTGTTGCTCCCGAGGAG

Hi sProThrLeuLeuThrGl uAl aProLeuAsnProLysAl aAsnArgGl uLysMetThrGl nIl eMetPhe- Exon. 2CACCCCACCCTGCTCACAGAGGCCCCGCTGAACCCCAAGGCCAACCGGGAGAAGATGACTCAGATCATGTTT

GluThrPheAsnValProAl aMetTyrValAl all eGI nAl aValLeuSerLeuTyrAlaSerGlyArgThrGAGACCTTCAATGTCCCTGCCATGTACGTGGCCATCCAGGCAGTGCTATCCCTGTATGCTTCTGGCCGTACC

ThrACAGGTATGCTGGGCTCTGGGGACAGTTACTGATGAATCACATTCCCAAGTCACCGACCTTGCTGTGAATCA

GATCCCCCAGTTGAAAAAGGGATAATCCCTTTCCTCCCATTCCCTAGCAAGGTCTGTGCTAAGAGAAAGAGT

TAACGGTAGTGCCCTGAGGTTAC6TTTCGGAGCACAATTATTATTGTTGAGCTGATAGCTTGTGGAGGTGGGC

CTTCCCTCATTTAAAGCTCAGCGCAGTGTAGCAGCTTGGAGTGCAGCAGTCATTGTTATGTGTTTAAACCAT

CACATCACCTGGGCAAGCATCCCCAAGGAGAATACATTCCATACAGGGTCTGACTCAAAAGAGAGAGAAACGIntron II

TGTAAGTTCAATAGGAGCAAAGAAAAACACCCTTGGGTGCTTACATAATGTGGCTGACAAGAAAGATGGTCA

TTTGAAAGTGTCCTCGGGAATTTTTTCTACTATAATAGTTAAAAAGATGAGCTGCAGCTTGCTTCAGATTTA

GTATTCCTGATGCGCATTTTTATTCTTTGTGTGTAAGGAATCTAATTTTATCTGGATCAATGCCCATTGCTA

GCATCTCTTAGCCAAGATTGGAAGCGGGCTTTGCCGTGGCTAGAGCAGTGGTGTTGTCCTCAGGAATTTACC

GlyIleValLeuAspSerGlyAspGlyValThrHisAsnValProIleTTGTTCTTGTGTACTTCCCCGGGCAGGCATTGTTCTGGACTCTGGGGATGGTGTAACTCACAATGTCCCCATC

TyrGl uGlyTyrAl aLeuProHi sAl all eMetArgLeuAspLeuAl aGlyArgAspLeuThrAspTyrLeuTATGAGGGCTACGCTTTGCCCCATGCCATCATGCGTCTGGATCTGGCTGGTCGGGACCTCACTGACTACCTC Exon 3

MetLysIl eLeuThrGl uArgGlyTyrSerPheVal ThrThrATGAAGATCCTCACTGAGCGTGGCTACTCCTTTGTCACCACTGGTGAGTGTGTGTGTCTCATCTGCCACAGT

GTGGGTCTGCTTTCCTCCTCTCTCACTGAATCCGCCTACCTCCCTATAATTGACTTCTTGCTTCAGAGCATG Intron III

Proc. Natl Acad. Sci. USA 79 (1982)

Al aGl uArGl ulleValArgAspIl eLysGi uLysLeuCysTyrValACTGTGATACTCTTTATTTCTGTAGCTGAACGTGAAATTGTCCGTGACATTAAAGAGAAGCTGTGCTATGTC

tAl aLeuAspPheGl UAsnGlu~ietAlaThrAlaAlaSerSerSerSerLeuGl uLysSerTyrGl uLeuProGCCCTGGATTTTGAGAATGAGATGGCCACAGCTGCCTCTTCCTCCTCCTTGGAGAAGAGCTATGAACTGCCT Exon 4

AspGlyGl nVal lIeThrIleGlyAsnGluArcqPheArgCysProGluThrLeuPheGl nProSerPhell eGATGGCCAAGTCATCACTATCGGCAATGAGCGCTTCCGCTGTCCTGAGACACTCTTCCAGCCCTCCTTCATT

GGTGAGTTGTAGGGTCTGGTGTAGAGGCACGATTTTCCTGGAAATCTTAGGGTCTCCCAGAGTAAAATCTAG

AATTCTCArGAAGCCCTTGAGTTAAAAGAAGTCATTGTTTGGATTCCCACACAGCTCA;ACCTCAAGTCCTGAIntron IV

AAAATAAAGGATGACAGAGAGTAGTAACTGAATAGCACTA'TCTGCAG-------------------------

CTGCAGTGTGTCTTATAGGGGAACATATGTTTCAGAGACAAATGGTGACAGCTCCCCCACACAAAGMGTTC

TGTTCTCTTCCCTCTACCTTGACCTGAATGCACTGTGATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG

GlyMetGl uSerAl aGlyIl eHi sGluThrThrTyrAsnSerll eTGTGTGTGTGTGTGTGACTCGTTCCCAGGTATGGAATCTGCTGGCATCCATGAAACAACTTACMTAGCATC

MetLysCysAspIleAspIIeArgLysAspLeuTyrAlaAsnAsnVal LeuSerGlyGlyThrThrMetTyrATGAAIGTGTGACATTGATATCCGCAAGGACCTGTATGCCAACAATGTCTTATCTGGAGGCACCACTATGTAC Exon 5

ProGlyll eAl aAspArgMetGl nLysGtuIl eThrAl aLeuAl aProSerThrMetLysll eLysCCTGGTATTGCTGATCGTATGCAGAAGGAAATCAC.TGCTCTGGCTCCTAGCACCATGAAGATTAAGGTAMG

AACTTTTGTGAGTGGGAGATCGAGGCAGGTCTTGGTATTCTMGCGAACTACGTTCCAAATTCCTTTTCCCT Intron V

TAGGTAAGTGGAGAGGTTCCATTTTAATAATAGAATATATCTTTMAATATATAMTGATGAAATAGAGGTGA

GTTCATATAACTTGATTGGCCATATTATTTCGTGGTATGACATATCCCACATTATAGCGAATTMTATCTAAT

GGTTTTTCTGTGAATCCTCCCMTGTGTTATTTGCTCCCTTGCTTGGAACTTCAGAGTTCACTGGMGTTTT

Ile leAl aProProGl uArgLysTyrSerValTrpIl eGlyGlySerIleLeuAlaTGTTTTCTTCTGCAGATTATTGCTCCCCCTGAGCGTAAATACTCTGTCTGGATTGGGGGCTCCATCTTGGCC

SerLe'iSerThrPheGl nGl nMetTrpIle.SerLysGl nGl uTyrAspGl uAl aGlyProSerIleValH1 s Exon 6TCTCTGTCCACCTTCCAGCAAATGTGGATTAGCAAGCAAGAGTACGATGAGGCAGGCCCATCCATTGTCCAC

ArgLysCysPheCGCAAATGCTTCTAAGATGCCTTCTCTCTCCATCTACCTTCCAGTCAGGATGACGGTATTATGCTTCTTGGA

GTCTCCCAAACCACCTTCCCTCATCTTTCATCAATCATTGTACAGTTTGTTTACACACGTGCAATTTGTTTG'

TGCTTCTAATATTTATTGCTTTATAAATAAACCAGACTAGGACTTGCAACCTATAAAAGCCTCTCGTTTGTT

TTTGGGGTAGGCGTGG6GTGGGGCAGG

FIG. 4. Nucleotide sequence of human cardiac muscle actin gene. The nucleotide sequence was determined by the strategy indicated in Fig.2C. The amino acid sequence of cardiac muscle actin determined by Vandekerckhove et al. (1) is shown above the nucleotide sequence. The verticalarrows under the nucleotide sequence show the splicing sites. The 3' polyadenylylation signal sequence and the poly[d(T-G)] stretch are underlined.

muscle actin gene detected more than 20 hybridizable bandsin the EcoRI-digested human DNA, including the 13-kb frag-ment of the cardiac muscle actin gene. This observation agreeswith the recent observation that there may be 20-30 actin oractin-like genes in the human genome (19). By considering thatonly six actin isoforms have been identified in the vertebrates,

the detection of 20 hybridizable bands suggests the presenceof pseudogenes or functional genes coding for unknown actinisoforms. On the other hand, there is a possibility that some ofthe 20-30 bands observed in the EcoRI blot may be due tohybridization to sequences unrelated to the actin gene sequencebecause our probe contains intervening sequences as well as

EcoRI EcoRI Xba I Hinfl Xba

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

13.0- _ -

.;.

a.l _-

I L

A

FIG. 5. Blot-hybridization analysis of cardiac muscleactin gene in human genome. AHa-25 DNA (1) (1-5 ng) orhumanDNA from HeLa (2) or Hut-14 (3) cells (20 gg) werecompletely digested with various restriction enzymes(shown), electrophoresed on agarose gels, and hybridizedto 32P-labeled Pst I 2.2-kb fragment (A), Pst I 0.4-kb frag-ment (B), orHinfI 0.3-kb fragment in the PstI 0.6-kb frag-ment (C). The locations of these fragments are shown inFig. 2 B and C. The hybridization was carried out as de-scribed (5). Filters were washed three times in 2 x NaCl/

- Cit containing 0.5% NaDodSO4 at 6500 and twice in 0.2 xC NaCl/Cit containing 0.5%o NDodSO4 at 5000.

2.1

1.5

0.7

kb

B

Proc. Natl. Acad. Sci. USA 79 (1982) 5905

coding sequences. It is noteworthy that in our previous studiespcHa-1 DNA, which contains a cDNA copy of human (3-actinmRNA, detected only two bands under the same hybridizationcondition (5).

(ii) On the other hand, the Pst I 0.4-kb fragment that wasderived from intron IV gave a single band, suggesting that thisintron sequence was not conserved in human actin gene familyor that it was never present in the ancestral actin gene but isa recent insertion element during the evolution of the cardiacmuscle actin gene. It is also likely that the human genome hasonly one or a few cardiac muscle actin genes. The same restric-tion pattern observed between chromosomal DNA and clonedDNA clearly indicates that no artificial recombination occurredduring cloning procedure.

(iii) When the 3' untranslated sequence of AHa-25 was usedas a probe, we detected two hybridized bands (Fig. 5C). The2.1-kb fragment contains cardiac muscle actin gene becausecloned DNA showed the same 2.1-kb fragment. The other hy-bridized band, the 1.5-kb fragment, is not derived from cyto-plasmic 3- or y-actin gene because the 3' untranslated regionof these genes showed no homology to that of cardiac muscleactin gene as described above. Shani et aL (27) have suggestedsubstantial sequence homology between the 3' untranslatedregion of rat skeletal and cardiac muscle actin gene. It is prob-able that the 1.5-kb fragment contains one of the other muscleactin genes (skeletal or smooth muscle).

(iv) No polymorphic region was detected between the twohuman cell lines.Our recombinant phages were isolated from a library of

cloned human DNA fragments derived from a single individual.No recombinants containing other types of actin genes wereisolated in spite of extensive screening. In contrast to our find-ing, the isolation of a number of actin or actin-like genes fromthe same library has been reported (19).Two explanations are possible for the difference between lab-

oratories in the number of the actin genes isolated. First, weused pcDd DNA which contains a cDNA copy ofDictyosteliumactin mRNA as a hybridization probe, whereas chicken or Dro-sophila actin genes were used by others. Previously, we foundthat pcDd DNA hybridized with cytoplasmic (3-actin mRNA butnot with y-actin mRNA and produced a single band in theEcoRI-digested human DNA under a stringent hybridizationcondition, whereas pcHa-1, which contains a cDNA copy ofhuman ,B-actin mRNA, hybridized with both 83- and y-actinmRNA and gave two bands in the EcoRI-digested human DNA(5). On the other hand, the 5' part of the cardiac muscle actincoding sequence produced more than 20 bands under the samehybridization condition. Second, the difference in the numberof actin genes isolated may be due to the difference in the strin-gency of the hybridization condition used for screening. Wehave noticed that under our less stringent condition, our probehybridized to additional DNA fragments in EcoRI-digested hu-man DNA. Recently, we have isolated at least seven actin oractin-like genes using pcDd as a probe under a less stringenthybridization condition but from a different DNA library thatwas made from a transformed human cell line, HuT-14, (un-published data). It is likely that DNA libraries ofdifferent originor preparation are composed of a different population of re-striction fragments.One of the interesting features of the structure of the human

cardiac muscle actin gene is the presence of a repetitive se-

quence, (dT-dG)25, next to the 3-splicing site of intron IV. Re-cent reports indicate that left-handed helical conformations areobserved with poly[d(A-C)d(G-T)] (28). Left-handed DNA hel-ices have so far been observed only with synthetic DNA poly-mer (29), and its biological implications have been unknown.Our finding of (dT-dG)25 in human cardiac muscle actin geneprovides evidence for the presence ofDNA sequences that mayform left-handed DNA helices in mammalian cells.We thank Dr. Tom Maniatis for providing us with a human DNA

library, Dr. Igor Dawid for his kind advice, and Dr. John Fagan for hiscritical review of the manuscript. H. H. is specially grateful to Dr. Hi-roaki Ohkubo for his abundant advice and suggestions and to Ms. Yuan-Ying Tsai Hamada for her help in screening the DNA library. Thanksare also given to Ms. Linda Nischan for her help in preparing themanuscript.

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Cell Biology: Hamada et al.