THE JOURNAL OF BIOLOGICAL CHEMISTRYVol. 257, No. 18, Issue of September 25, pp. 11078-11086, 1982Printed in U.S.A.

Cloning and Characterization of cDNA Sequences Corresponding toMyosin Light Chains 1, 2, and 3, Troponin-C, Troponin-T,a-Tropomyosin, and a-Actin*

(Received for publication, February 16, 1982)

Leonard I. Garfinkel, Muthu Periasamy*, and Bernardo Nadal-Ginardt§From the Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461

A library of cDNA clones was constructed from adultrat skeletal muscle mRNA, from which a set of contrac-tile protein clones was selected. These clones wereidentified by sequencing the cDNA inserts and compar-ing the derived amino acid sequences with publishedsequences of rabbit contractile proteins. In this man-ner, clones corresponding to myosin light chains 1, 2,and 3, troponin-C, troponin-T, a-tropomyosin, and a-actin were identified. A high degree of amino acidsequence conservation was found upon comparison ofthe rat and rabbit proteins.

Using the cDNA clone panel, we analyzed the expres-sion of abundant rat muscle mRNAs. We show thatabundant rat muscle mRNAs can be classified into fourdevelopmentally regulated groups, based upon theirexpression at different stages of myogenesis. One classof mRNAs is expressed during all stages of muscledevelopment. Since these mRNAs are also present innonmuscle tissues, we conclude that they code forhousekeeping proteins. The second class of mRNAs ispresent in both embryonic and adult muscle, while athird class of mRNAs is expressed only in adult muscle.A small number of mRNAs, which are present atgreater levels in undifferentiated myoblasts than inadult muscle, comprise a fourth class. These resultssuggest the existence of at least four modes of genecontrol during myogenesis.

Terminal differentiation of muscle cells is a complex processwhich is characterized by dramatic alterations, both biochem-ical and morphological, in the cellular phenotype. Alignmentof mononucleate myoblasts and subsequent fusion of theirplasma membranes to form multinucleate myotubes is accom-panied by synthesis of vast quantities of muscle-specific pro-teins (for review, see Ref. 1). Among these are the proteins ofthe thin and thick filaments of the contractile apparatus:myosin heavy chain, three myosin light chains, three tropo-nins, two tropomyosins, and a-actin. Devlin and Emerson (2)have shown that these proteins accumulate in a coordinatemanner in developing quail muscle: i.e. their accumulationbegins at the same time, they have similar synthetic rates, and

* This work was supported by grants from the National Institutesof Health, the New York Heart Association, and the Muscular Dys-trophy Association of America. The costs of publication of this articlewere defrayed in part by the payment of page charges. This articlemust therefore be hereby marked "advertisement" in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

t Present address, Department of Pediatrics, Harvard MedicalSchool and Department of Cardiology, Children's Hospital MedicalCenter, 300 Longwood Ave., Boston, MA 02115.

§ To whom all correspondence and requests for reprints should beaddressed.

they reach their steady state levels at the same time. More-over, translatable mRNAs coding for these proteins also ac-cumulate coordinately (3). In rat muscle, steady state levelsof mRNAs coding for myosin heavy chain, myosin light chain2, and a-actin increase in parallel with the levels of theirrespective proteins (4-7).

The elucidation of the molecular mechanisms responsiblefor the coordinate expression of the contractile proteins re-quires an analysis of the metabolism of their respectivemRNAs. Specifically, it must be determined how syntheticrates and stabilities of individual mRNAs ultimately affecttheir final steady state levels during muscle development. Forthese studies, it is necessary to have specific cDNA hybridi-zation probes. To this end, we have constructed a library ofrat muscle cDNA clones from which a set of seven contractileprotein clones was selected. In this paper, we report thecharacterization of clones coding for a-actin, myosin lightchain 2, myosin light chain 1 or 3, troponin-C, troponin-T, anda-tropomyosin. The characterization of an adult myosin heavychain cDNA clone will be reported elsewhere.'

Analysis of the entire cDNA library and a large number ofindividual clones shows that there are four developmentallyregulated classes of abundant mRNAs in skeletal muscle. Oneclass of mRNA is expressed at all stages of muscle develop-ment, and is even present in undifferentiated myoblasts. ThesemRNAs are also present in nonmuscle tissues, and are likelyto code for housekeeping proteins. A second class, comprisingthe majority of abundant muscle mRNAs, is expressed inmyogenic tissue beginning in early embryogenesis and contin-uing into adulthood. A third class is expressed in adult tissue,but not in early embryonic muscle. A fourth class consists ofmRNAs that are present in undifferentiated myoblasts, butonly at greatly reduced levels during later stages of muscledevelopment.

EXPERIMENTAL PROCEDURES

Preparation of RNA-Total RNA was extracted from hind legmuscle of adult rats by the hot phenol procedure (8). To eliminateDNA, the RNA was precipitated once with guanidine hydrochloride(9) and ethanol. Total cytoplasmic RNA was isolated from L6E9myoblasts and myotubes as described (4). Poly(A+) RNA was purifiedfrom total RNA by affinity chromatography on oligo(dT)-cellulose(Collaborative Research) (10). This fraction, which represented 3.5%of total skeletal muscle RNA and approximately 2% of total L6E9RNA, was ethanol-precipitated before further use.

Preparation of Single-stranded cDNA-cDNA was prepared in a200-pl reaction containing 100 mM Tris, pH 8.3, 5 mM MgC 2, 10 mMdithiothreitol, 1 mM each of dGTP, dATP, and dTTP (Schwarz/Mann), oligo(dT)12-18 (P-L Biochemicals), 0.1 mM [3H]dCTP (Amer-sham Corp., 20 Ci/mmol), 50 jig of poly(A+ ) RNA, and 440 units ofavian myeloblastosis virus reverse transcriptase (generously providedby Dr. J. W. Beard, Life Sciences, Inc.). The RNA was dissolved in

'D. Hornig and B. Nadal-Ginard, manuscript in preparation.

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16, does have a poly(A) region and, based on their restrictionmap, we conclude that pAC-16 also has such a poly(A) tract.The a-tropomyosin clone, pATM-6, is a fragment from themiddle of the mRNA and has no poly(A) tract. In the followingparagraphs, the clones are presented in order of increasingsize of the mRNAs for which they code.

Myosin Light Chain 2-pLC2-18 (Fig. 3) contains se-quences homologous to fast muscle myosin light chain 2mRNA (27). The 500-base pair cDNA insert contains approx-imately 450 bases of mRNA sequence which represents 50%of the mRNA. The clone codes for 102 out of 169 amino acids,or 60% of the protein. In addition, the clone contains a 3'nontranslated region of approximately 100 bases, and apoly(A) tract of approximately 50 bases.

Troponin-C-pTNC-45 (Fig. 4) contains sequences homol-ogous to fast muscle troponin-C (28). The 500-base pair insertcorresponds to about 450 bases of troponin-C mRNA, about50% of the message length, including a poly(A) tract of about30 bases (not shown). The insert contains enough sequence tocode for 88 amino acids, beginning with residue 75, andcontinuing to the end of the protein. This constitutes about55% of the protein. In addition, the clone contains a non-translated region of about 150 bases (not shown).

Myosin Light Chain 1/3-pLC-84 (Fig. 5) may code foreither fast muscle myosin light chain 1 or 3; the amino acidsequence that we derived is present in both proteins (29, 30).The cDNA insert contains sequences coding for 136 aminoacids which represents 72% of light chain 1 (190 amino acids)or 92% of light chain 3 (149 amino acids). In addition, pLC-84contains a 3' nontranslated region of approximately 275 basesand a poly(A) tract of 41 bases. The structure of the cDNAinsert is interesting in that the right-hand Pst I/Eco RIfragment is an inverted repeat of part of the left-hand Pst I/Eco RI fragment. The smaller of the two fragments is in thecorrect orientation with respect to the translated region, whichis entirely contained within the middle Eco RI fragment.However, it is the larger fragment, generated artifactually,that contains the poly(A) tract. The sequences of the twofragments are co-linear for 152 bases, the entire length of thesmaller fragment. This type of phenomenon has been reportedpreviously, and possible mechanisms for the generation ofsuch artifacts have been suggested (31).

Troponin-T--pTNT-15 (Fig. 6) corresponds to fast muscletroponin-T (32). The cDNA insert contains approximately 950bases homologous to troponin-T mRNA, including a poly(A)tract of about 30 bases. Thus, pTNT-15 represents 80% of theentire mRNA sequence. The clone contains a 3' nontranslated

PstI XbaI EcoRI AvaIa. I I I I

lOObp

11081

Hind XboI

I . -§ .I

55 60 70b. ATT CTC CTC TTC GAC AGA ACC GGT GMAA TGC AAG ATC ACC TTA AGT CAG GTG GGC GAC GTC

C. I L L F T E C K I T L S Q V G D VF r D S

80 90CTC CGG GCT CTG GGC ACC AAT CCC ACA AAT GCA GAA GTC AAG AAG GTT CTC GGG AAC CCT

L R A L T N P T N A E V K X V L G N P

100 110AGC AAT GAA GAG ATG AAT GCT AAG AAA ATC GAG mTT GAA CAG mTT CTG CCC ATG ATG CAAS N KE E M N A K K I E F E Q P FL P Q

D Q L

120 130GCC ATC TCC AAC AAC AAG GAC CAG GGA GGC TAT GAA GAT TTC GTT GAG GGT CTG CGT GTCA I S N N K D Q G G Y E D F V E G L R V

140 150TTC AC AAG GAG GGC ART GGC ACC GTC ATG GGT GCT GAG CTC CGC CAT GTC CTC GCC

PF D K E N G T V M G A E L R H V L AD - G

160 170ATC CTG GGA GAG AAG ATG AAG GAG GAG GAG GTA GAA GCA TTG CTG GCG GGC CAG GAG GAC

T L G E K M K E E E V E A 5 A G Q D

180 190TCC ART GGC TGC ATC AAC TAT GAA GCT mTT GTG AAA CAC ATC ATG TCT GTC TAA AACGAGA

S N G C E A F V K A I t S V ed

I

ATTCAAGAAMGMCACAGTGTTGGGGGACTGGCCAGAAAGTCAGTTCAAGAACACCTATGGCTAACTGTCAACACCAGCTT

GCCACCACCCGGAAGAACAAACAATCTAGACCATTCTAGATTGAAGGATT C CTGAAGTTTTATCAACCTTGAGTTTTT

CATGGCACACACATCAGGTTGATTCTCMGTCCATGTACCACCTTTATGATCTACTTGGGTCCACAACATTCATMG

AGCACTACATCAATAMCTTGGTCAGTCTGGAAGACTC (A )41

FIG. 5. Restriction map and sequence data of pLC-84(myosin light chain 1/3). pLC-84 DNA was digested with Eco RI,5' end-labeled, and redigested with Pst I. Both Eco RI/Pst I fragmentswere sequenced directly. The middle Eco RI fragment was strand-separated before sequencing. Alternatively, the plasmid wasdigested with Pst I, 3' end-labeled, and redigested with Eco RI. ThePst I/Eco RI fragments were sequenced after purification. The un-derlined portion of the nontranslated region represents sequences ofthe inverted repeat fragment in the correct orientation and position(see text). The AATAAA polyadenylation site is underlined. Num-bers above codons refer to rabbit myosin light chain 1. a, restrictionmap of pLC-82; b, rat myosin light chain 1/3 cDNA sequence; c, ratand rabbit myosin light chain 1/3 amino acid sequences.

Pst I KpnI Kpn Ia. I I - I

Pst II

100 bpk----

75 80 90b. TTT GAA GAG TTC TTG GTC ATG ATG GTG CGC CAG ATG AAA GAG GAT GCG AAA GGG AG AGC

C. F E E F L V M M V R Q M K E D A K G K S

100 110GAA G GAGAA CTG GCT GAG TGT TTC CGC ATC TTT GAC AGG AAC GCA GAC GGT TAC ATT GAT

E E E L A E C F R I F D R N A D G Y I D

119GCC GAG GAG CTG GCT

A E E L A

FIG. 4. Restriction map and sequence data of pTNC-45 (tro-ponin-C). pTNC-45 DNA was digested with Pst I, 3' end-labeled,and redigested with Kpn I. The smaller Pst I/Kpn I fragment wassequenced. a, restriction map of pTNC-45; b, rat troponin-C cDNAsequence; c, rat troponin-C amino acid sequence. The entire rattroponin-C amino acid sequence presented is identical to the corre-sponding rabbit sequence.

region of approximately 140 bases, leaving 780 bases of trans-lated sequence, enough to code for the entire 259 amino acidprotein.

a- Tropomyosin-The amino acid sequence of the region ofpATM-6 that we examined (Fig. 7) is 100% homologous toboth rabbit a- and 83-tropomyosins (33, 34). Moreover, rabbitcardiac and skeletal a-tropomyosin have identical amino acidsequences (34), and may be coded by the same mRNAs.However, the mRNAs coding for a- and /f-tropomyosins donot cross-hybridize at high stringency (35). Therefore, onewould expect that an a-tropomyosin clone, but not one con-taining /f-tropomyosin sequences, would hybridize to cardiacRNA, since heart tissue contains only a-tropomyosin (36).When pATM-6 was used to probe size-fractionated rat cardiacRNA in a blot hybridization, a strong signal was produced,even after stringent washes in 0.1 x SSC at 70 °C (not shown).We deduce, therefore, that pATM-6 corresponds to a-tropo-myosin. The 350-base pair insert contains about 300 bases oftranslated sequence, enough to code for 100 amino acids out

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Pst II

Ava II

PstI XbaI PstII I I

100 bp_

99 110b. AAG CGC CGT GCA GAG AGG GCT GAG CAG CAA AGG ATT CGC GCT GAG AAG GAG CGG GAG CGCC. X R A A E R A E Q Q R I R A E K E R E R

120 130CAG AAC AGA CTG GCG GAG GAG AAG GCC AGA AGA GAG GAG GAA GAT GCC AAG AGG AGA GCT

Q N R L A E X A R E E E D A K R R A

140 150GAA GAT GAC TTG AAG AAG AAA AAG GCT CTG TCC TCT ATG GGT GCC AAC TAC AGC AGC TAC

D D L K K K K A L S S M G A N Y S S Y

g

160 171CTG GCC AAG GTT GAC CAG AAG AGA GGC AAG AAA CAG ACG

L A K V D Q K R G K K Q T

A

175 180ATG AAA AAG G ATT CTT

M K K K I L

190 200GCT GAA AGG CGC AAA CCT CTG AAC ATT GAC CAT CTT AGT GT GAT AAG CTG AGG GAC AAG

A E R R K P L N I D H L S D D K L R D K

E

210 220GCC AAG GAA CTC TGG GAT ACC TTG TAC CAA CTT GAA ACT GAC AAA Tml GAG TTT GGG GAG

A K L W D T Y Q L E T D K F F G E

230 240AAG CTG AAA CGT CAG AAA TAC GAT ATC ATG AAC GTC CGG GCC AGG GTG GAG ATG CTG GCC

K L K R Q K Y D I M V R A R V E M L A

250 259AAG TTC AGC AAG AAG GCC GGT GCC ACG GC AAG GGC AAA GTC C GGG CGC AAG TGG AAG

K F K K A A T A K K V C C R K W K

T

TAA ACGAGGTGCCTGCAGCAGAGACCATCAACCCTGATCTTGCCAAGGTCCCTGCCGCTATAACTTATTACTTTGCCATCCCACCAGCTCTGTAT

end

TCTCCAACCCTCATGCCCAAGCACTTTTGGGAACTCAGGGCCCACCCTGCTGCAATGTCCTCGCCCTCTGGGTCTAGA

AATAAAGTTATCAGACTCCC (A)n

FIG. 6. Restriction map and sequence data of pTNT-15 (tro-ponin-T). pTNT-15 DNA was digested with Ava I, 5' end-labeled,and redigested with Pst I. The two Ava I/Pst I fragments weresequenced from the Ava I site. The small Pst I fragment was 3' end-labeled, and strand-separated before sequencing. The AATAAA pol-yadenylation site is underlined. a, restriction map of pTNT-15; b, rattroponin-T cDNA sequence; c, rat and rabbit troponin-T amino acidsequences.

Pst II

Pst II

RNA was probed, it hybridized to a band corresponding insize to ,/y-actin mRNA. In addition, in vitro translation ofskeletal muscle mRNA purified by hybridization to filter-bound pAC-16 plasmid DNA produced a band that co-mi-grated with skeletal muscle actin on sodium dodecyl sulfate-polyacrylamide gels (not shown). pAC-16 was positively iden-tified as a-actin by sequencing a restriction fragment that wepredicted should contain part of the 3' nontranslated region,based on the restriction map of Shani et al. (26). The sequenceof this fragment is identical to positions 49 through 111 of thenontranslated region in the published sequence (26). It hasbeen shown that the mRNAs coding for several eucaryoticproteins from different tissues or developmental stages showgreater sequence divergence in their 3' nontranslated regionsthan in their translated regions (26, 37, 38). Our finding ofcomplete sequence homology in a fragment from the 3' non-translated regions indicates that pAC-16 contains a actin se-quences.

The identities and other characteristics of these six clonesare summarized in Table II. Analysis of tissue specificity ofeach of these six clones shows that all but one code forembryonic/adult mRNAs, i.e. those that are present in L6E9myotubes and adult skeletal muscle only. The exception, pLC-84, codes for adult myosin light chain 1 or 3, neither of whichis expressed in differentiated LE 9 myotubes. Although pAC-16 codes for a-actin, an embryonic/adult mRNA, it also hy-bridizes the fi/y-actin mRNA, which is present in myoblastsbut not in differentiated muscle. This pattern of hybridizationsexplains the relative signal intensities of those clones in Fig.1 marked by vertical arrows. One of them is pAC-16 and theother is similar to pAC-16, but with a shorter insert. Thehybridization signal is greater when adult skeletal musclemRNA is used as probe than when myoblast mRNA is used.This can be explained in two ways: 1) actin mRNA is moreabundant in adult skeletal muscle than in myoblasts, or 2) the

AluI BamHIPst I BglI TaI TaqiAluI

a.TaqI PstI Pst I

I I I

!oobIi

Pst II

100 bpI----

93 100 110b. CAG CTG GTT GAG GAG GAG TTG GAT CGC GCC CAG GAG CGT CTG GCC ACA GCT CTA CAG AAG

C. Q L V E E E L O R A A Q E R A Q K

120CTG GAG GAG GCT GAG AAG GCT GCX

L E A K A A

FIG. 7. Restriction map and sequence data of pATM-6 (a-tropomyosin). pATM-6 DNA was digested with Pst I and 3' end-labeled, and the fragments were purified. The smaller fragment wasstrand-separated and sequenced from the internal Pst I site. a, re-striction map of pATM-6; b, rat a-tropomyosin cDNA sequence; c,rat a-tropomyosin amino acid sequence. The entire rat a-tropomyosinamino acid sequence presented is identical to the corresponding rabbitsequence.

of a total of 284, or 35% of the protein. The cDNA insertrepresents about 20% of the 1350-base a-tropomyosin mRNA.

a-Actin-pAC-16 (Fig. 8) was preliminarily identified as a-actin, based on a determination of the size of the mRNA towhich it corresponds. The clone hybridized to a mRNA thesize of a-actin mRNA in blot hybridizations with skeletalmuscle RNA. However, when myoblast or smooth muscle

b. GACCATCGTGCTATGGTTGCAGGGTGGCCCCATCCTCCGCCGTGGCTCCATCGCCGCCACTG

FIG. 8. Restriction map and sequence data of pAC-16 (a-actin). pAC-16 DNA was digested with Taq I, 5' end-labeled, andredigested with Pst I. The smaller Taq I/Pst I fragment was purifiedand sequenced. The sequence corresponds to positions 49 to 111 ofthe 3' nontranslated region (26). a, restriction map of pAC-16; b, DNAsequence of the small Taq I/Pst I fragment of pAC-16.

TABLE II

Identities and characteristics of cDNA clones

All of the contractile protein clones, except pLC-84, are in thevector pBR322. pLC-84 was cloned in pBR325.

Insert

Clone Size ClassProtein

basepairs

pLC2-18 500 Embryonic/adult muscle Myosin light chain 2pTNC-45 500 Embryonic/adult muscle Troponin-CpLC-84 900 Adult muscle-specific Myosin light chain

1/3pTNT-15 1000 Embryonic/adult muscle Troponin-TpATM-6 350 Embryonic/adult muscle a-TropomyosinpAC-16 700 Embryonic/adult musclea a-Actin

a Hybridizes to /¥-actin in LE 9 myoblasts (see text).

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TABLE IV

Relative coding sequence content of contractile protein mRNAsmRNA coding length was determined by multiplying the protein

length (in amino acids) by 3. The length of the 3' nontranslated regionwas determined by locating the protein termination codon and thepoly(A) tract on the clone and calculation of the number of interven-ing bases. The length of the 3' noncoding region of a-actin mRNAwas reported previously (26). mRNA total length was calculated bymeasuring relative mobility of each mRNA on RNA blots and plottingits size on a graph of RF versus size in bases using as size markersglobin mRNA, and 18 and 28 S ribosomal RNA (1900 and 5200 bases,respectively, Ref. 66). Proteins are listed in order of increasing size.

Protein mRNA 3' non mRNACoding/Protein coding tnon- Codingtalding tol totallength length coding length total

amino basesacids

Myosin light chain 3 149 447 275 940 0.48Troponin-C 159 477 150 920 0.52Myosin light chain 2 169 507 100 880 0.58Myosin light chain 1 190 570 275 1150 0.50Troponin-T 259 777 140 1200 0.65a-Tropomyosin 284 852 1350 0.63a-Actin 374 1122 240 1650 0.74

smaller mRNAs is translated. This difference could be due tolonger poly(A) tails in the smaller mRNAs, or simply torelatively more nontranslated sequence. However, a survey ofthe lengths of the 3' nontranslated regions, as shown in TableIV, indicates that the latter possibility is more likely. Theaverage length of the 3' nontranslated regions of a-actin andtroponin-T mRNAs is 190 bases, while the average length ofthose regions in mRNAs coding for myosin light chain 2,myosin light chain 1/3, and troponin-C is about 175 bases.Thus, the smaller mRNAs with short coding regions have 3'nontranslated regions approximately equal in length to thoseof the larger mRNAs with longer coding regions. Therefore,the smaller mRNAs have a greater amount of 3' nontranslatedsequence relative to their coding capacity than do the largermRNA's.

A feature that at least two of the contractile protein mRNAshave in common with several other eucaryotic mRNAs is thepresence near the poly(A) tail of the sequence AAUAAA. ThemRNA coding for myosin light chain 1/3 contains this se-quence in the expected position, beginning 27 bases from thepoly(A) tail (Fig. 5), and mRNA coding for troponin-T hasthis sequence beginning 20 bases from the poly(A) tail (Fig.6). This sequence has been found in several mRNAs studied(44), and has been shown to form part of the recognition sitefor polyadenylation of late SV40 mRNAs (45). Preliminarydata indicate that both troponin-C and troponin-T mRNAhave this sequence. However, this finding must be verified byfurther sequencing. The 3' nontranslated region of a-actinmRNA contains the similar sequence AUUAAA (26).

DISCUSSION

The aim of this work was to construct a library of rat musclecDNA clones, a large proportion of which would containsequences homologous to contractile protein mRNAs. It hasbeen shown (3-7, 46) that concurrent with the dramatic in-crease in the abundance of contractile proteins during muscledifferentiation, there occurs a comparable increase in the levelof mRNAs coding for these proteins. This suggests that thehighly abundant mRNA fraction whose appearance accom-panies muscle differentiation contains predominantly contrac-tile protein mRNAs. Paterson and Bishop (47) have foundthat 20% of chick embryo muscle mRNA consists of abundantsequences. Affara et al. (48) obtained nearly identical resultswith mRNA from mouse embryonal carcinoma-derived my-otubes. In light of these data, the finding that about 20% of

our library is composed of clones coding for abundant mRNAs,as demonstrated by colony hybridizations, shows that thecDNA population which we cloned resembles the mRNAstarting material with respect to abundance classes, a neces-sity in achieving our goal.

We believe that the four classes of mRNA to which ourcDNA clones correspond represent four developmentally reg-ulated groups of genes. The housekeeping class of mRNAsprobably code for so-called housekeeping proteins, since theyare equally abundant in all tissues tested, including smoothmuscle and fibroblast, pituitary, and hepatoma cell lines.2

Embryonic/adult muscle mRNAs code for muscle proteinswhose expression begins early in muscle development, whileadult-specific muscle mRNAs code for muscle proteins ex-pressed only later in development. Myoblast-specific mRNAscode for proteins whose abundance decreases during muscledifferentiation. That adult-specific muscle mRNA expressionbegins at some time after that of embryonic/adult mRNAs issupported by the finding that the L6 cell line (49), from whichL6E 9 was subcloned, contains a unique, embryonic form ofmyosin light chain 1, and shows no expression of adult formsof myosin light chain 1 or 3 (50, 51), coded by pLC-84, anadult-specific muscle clone. Furthermore, L6 contains an em-bryonic form of myosin heavy chain, rather than neonatal oradult forms (52). The L6E9 cell line, therefore, probably rep-resents early embryonic muscle, in which adult-specific musclemRNAs are not yet expressed. Zevin-Sonkin and Yaffe (53)have described a population of mRNA in adult rat skeletalmuscle, comprising 20% of abundant sequences, which doesnot hybridize to cDNA from fused L muscle cells, a lineclosely related to L6E9. Adult-specific muscle clones, whichrepresent approximately 25% of abundant sequences in ourlibrary, are probably derived from this adult-specific mRNApopulation. The existence of myoblast-specific mRNAs hasbeen suggested by previous studies. RNA population studies(54) have demonstrated a decrease in transcriptional diversityduring embryogenesis. More recently, in vitro translation ofmyoblast and myotube mRNAs and subsequent two-dimen-sional gel analysis of the protein products has demonstratedthe disappearance of several myoblast mRNAs during muscledifferentiation (46). Moreover, Ordahl et al. (55) have de-scribed the isolation of a cDNA clone whose correspondingmRNA is present only in myogenic tissue during early embry-ogenesis. Our myoblast-specific clones are being studied fur-ther in order to clarify some of their properties.

It is possible that there are actually more than four mRNAclasses in muscle. The figure that we present is, however, thelower limit. In fact, adult-specific muscle mRNAs can bedivided into two subclasses based on the expression of myosinlight chains 1 and 3 in developing muscle. Light chain 1mRNA begins to accumulate earlier in development than doeslight chain 3 mRNA.3 Therefore, adult-specific musclemRNAs can be subgrouped according to whether they beginto accumulate in middle or late embryogenesis.

The degree of amino acid sequence homology between ratand rabbit contractile proteins is consistent with the highlyconserved nature of these proteins throughout evolution. Thisconservation most probably reflects functional constraints onthese proteins that must be maintained in order to produce afunctional sarcomere. We have recently shown that for myosinheavy chain, there is a high level of nucleotide sequenceconservation from lower invertebrates to man (56-58). 4

2 L. Garfinkel, unpublished results. See also Fig. 1.3 L. Garfinkel, R. Gubits, and B. Nadal-Ginard, manuscript in

preparation.4 R. Wydro, H. Nguyen, R. Gubits, and B. Nadal-Ginard, submitted

for publication.

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Whether similar evolutionary constraints exist at the DNAlevel in genes coding for the proteins of the thin filamentremains to be determined. If the genes coding for the thinfilament proteins are as conserved as the myosin heavy chaingenes, they will provide an interesting system in which tostudy the evolution of the pattern of gene organization inmetazoan organisms.

There are at least three forms of each troponin and myosinlight chain: fast, slow, and cardiac (59-61), which are found,respectively, in fast and slow muscle fibers (62), and in cardiactissue. Slow and cardiac troponin-C have identical amino acidsequences (63), although they are not necessarily coded bythe same gene. Although it has not formally been classified asa fast or slow isozyme, a-tropomyosin is found in greaterabundance in fast muscle than slow muscle (36), and maysimply be the "fast" form of tropomyosin, f-tropomyosinbeing the "slow" form. This relationship is consistent with thehigh degree of homology between a- and /f-tropomyosin; of284 amino acids, they differ at only 39 residues (34). This isreminiscent of the high degree of homology between the slowand fast forms of the troponins (63, 64). The situation witha-actin is more complicated. There are many different genesfor actin in several organisms (65-67), and designations suchas fast or slow may be meaningless for this protein. It is ofinterest that pLC2-18 (myosin light chain 2), pLC-84 (myosinlight chain 1/3), pTNT-15 (troponin-T), and pTNC-45 (tro-ponin-C) all code for the fast forms of their respective proteins.One would expect a mixture of cDNA clones corresponding tothe slow and fast isozymes since leg muscle, the source ofmRNA for these cDNA clones, is a mixture of fast and slowfibers. The reason for this bias toward the fast forms of musclesequences is not known at this point, but provided that themRNAs for the fast and slow forms of these proteins show aminimal degree of homology, it will be relatively easy toisolate and analyze cDNA clones corresponding to the slowmuscle mRNA sequences.

The role of the 3' nontranslated region of mRNA as well asthe factors determining its length is unknown. The 3' non-translated regions of the mRNAs coding for the proteins ofthe thick and thin filaments, described in this paper, havesome interesting features worth mentioning, although theirbiological significance remains to be elucidated. There is noclear relationship between the length of the 3' nontranslatedregion and protein or mRNA size. In fact, a- and ,/y-actin aresimilarly sized proteins, yet their mRNAs differ in length byabout 550 bases (26). It is clear that the length of this regionhas no effect on translational efficiency, since myosin lightchain 1 and troponin-C have nearly identical synthetic ratesin muscle (2, 68), although the former has a 3' nontranslatedregion nearly twice as long as the latter.

The proteins of the contractile apparatus comprise a groupwhose synthesis and accumulation is coordinately regulated.The available data also suggest that their mRNAs accumulatein a coordinate manner (3-7).5 Except for fast myosin lightchains 1 and 3, which are not expressed in early embryonicmuscle, all of the contractile protein mRNAs studied are inthe embryonic/adult class. This suggests that at the veryleast, a subgroup of genes coding for embryonic/adult mRNAsis coordinately expressed. The existence of mRNAs that canbe grouped according to their pattern of expression into thefour classes described above raises significant questions abouttheir regulation. In order to understand the molecular basis ofgene regulation during differentiation, it is important to de-termine whether genes that code for a specific class of coor-dinately regulated mRNAs are subject to common regulatory

5 L. Garfinkel and B. Nadal-Ginard, manuscript in preparation.

mechanisms, different from the mechanisms operating in theexpression of genes belonging to other classes, or whethereach gene, regardless of class, reaches its characteristic devel-opmental and steady state pattern of expression in an individ-ual and specific manner. If the first possibility is operating, itwould be expected that genes belonging to one class will usethe same mechanisms of induction, transcriptional or post-transcriptional, and that all members of the group will exhibitsimilar transcriptional rates and half-lives of the mRNAswhich will, in turn, determine their pattern of accumulationand final steady state levels. If each gene is independentlyregulated, regardless of class, it is expected that the genesbelonging to one class will reach their characteristic develop-mental and steady state pattern of expression by widelydifferent mechanisms. The recombinant cDNA clones pre-sented here make it possible to directly measure the differentparameters involved in the expression of several mRNAs withat least four different patterns of expression. Data obtainedwith these clones should provide interesting insights into thebasic aspects of gene expression during cell differentiation.

Acknowledgments-The authors would like to acknowledge theexcellent technical assistance of Eva Bekesi. We thank Dr. BarbaraBirshtein for helpful discussions.

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L I Garfinkel, M Periasamy and B Nadal-Ginard3, troponin-C, troponin-T, alpha-tropomyosin, and alpha-actin.

Cloning and characterization of cDNA sequences corresponding to myosin light chains 1, 2, and

1982, 257:11078-11086.J. Biol. Chem. 

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