Eur. J. Biochem. 82. 601 -608 (1978)

Quantification of Myosin Heavy-Chain mRNA during Myogenesis JeHrey ROBBINS and Stuart M . HEYWOOD

University 01’ Connecticut Genetics and Ccll Biology Section, Storrs, Connecticut

(Received Jmuary 26 :September 9. 1977)

Complementary DNA (cDNA) was synthesized from a 26-S poly(A)-containing RNA fraction consisting predominately of myosin mRNA, and used to quantitate the amounts of myosin mRNA during development. RNA . cDNAmyolin hybridizations were performed with RNA isolated from tertiary cultures of chick fibroblasts, and the proportion of myosin RNA determined. The levels of myosin RNA present in primary chick myoblast cultures undergoing development were also analyzed by RNA . cDNAmYmin hybridizations. Correlation of these values with the rate of myosin heavy chain synthesis indicates that myosin mRNA is subject to post-transcriptional or translational control.

The biochemical and morphological events that characterize myogcnesis have bccn well documented [ 1 - 31. Terminal ditrerentiation is characterized by the formation of multinuclcated myotubes, which arise from thc fusion of the single myoblasts [4]. Cell I‘usion is closely followed by thc increased pro- duction of the cell-specialized proteins needed for the formation of thc contractile apparatus [l, 21. The heavy and light chains of myosin and other myofi- brillar proteins, such as actin. tropomyosin and the troponins, are synthesized and subsequently assembled into the sarconieric structurc.

This developmental process can be studied in cell culture. Myoblasts, obtained from 1 1-day chick embryonic leg muscle. will undergo terminal differen- tiation when cultured under suitable conditions [5 - 81. Examination of the multinucleated myotubes reveals a well-formed contractile apparatus and T-tubule system. and spontaneous contraction of myotubes can be observed [2,9].

Single myoblasts gegin to fuse 22-30 h after the initial plating. Subsequent myotube formation is rapid and the process is essentially complete within 24 h [lo]. The low levels of the myosin heavy chain present in the myoblast and early myotubes increase rapidly. Eventually myosin accounts for 7- 15% of the total protein synthesized in the fully differentiated myotubes [9,11]. Originally it was thought that the process of fusion may be a prerequisite for the syn- thesis of this protein [ l , 6,12 - 141. However, more

. ~ h h W I ’ i t / / f f J i i . I Icpcs. 44 2-hydroxyet1iyl)-1 -pipcrazineethanesul- phonicacid.

Dc/iuirioii. r,,! values are given in mol I ’ s; roil 2 is that value of )-<,/ at which the hall-maximal level of wiuration is reached. This V;IIIIC i b il characteristic constant o f thc rcaction.

recent experiments, showing large amounts of myosin in fusion-arrested cultures of myoblasts, have de- monstrated that formation of the myotubes is not required I‘or the synthesis of the myosin heavy chain [8 ,9 ,11,15] . Thus, fusion, while serving as a convenient developmental marker, is not necessarily the event that triggers terminal differentiation.

The rapid accumulation of’ myosin observed during myogenesis has prompted investigation of the control mechanisms underlying the process. A number of different experimental approaches have indicated that myosin synthesis may be regulated at the post- transcriptional level [16- 191. With the isolation and characterization of myosin mRNA [10,20] it became possible to demonstrate that a preparation of specific initiation factors could effect the translation of the messenger in r’itro in a heterologous cell-free system [18,21].

Buckingham e t (I/. [ 16,171, utilizing primary myo- blast cultures derived from fetal calf muscle, reported that the half-life of the 2 6 3 mRNA increases from 10 h in the myoblasts to over 50 h in the fully dif- ferentiated myotubes. In the myoblasts a large pro- portion of the 26-S mRNA could be isolated as translationally inactive messenger ribonucleoprotein particles. These authors report that the bulk of this population is eventually degraded, and is not utilized by the protein synthetic apparatus. The more stable 26-S mRNA present in the postfusion cultures can also be isolated in the RNA . protein form. However. in contrast to the 26-S mRNA isolated from earlier cultures, this long-lived species could later be found in the heavy polysomes. Apparently, during its syn- thesis and accumulation myosin mRNA can be tem- porally sequestered from its activation as a messenger and entrance into the polysomes.

hl I2

Heywood ol. [ 191 have isol:ited translatable niyosin niRNA froin the SO- 120-S region of poly- soiiies derived from I2-clny embryonic chick leg muscle. The associiition with the messenger ribonu- clciir protein pnrticlc fraction of ;I small RNA (trans- liltionill control KNA). which is able to inhibit the tr;insliition iii rim) o f the myosin messenger. has led to the forinulation of ;I model for translational activu- tion of the stored niyosin message [ 18.22.231.

In order t o elucidatc tlic role that post-transcrip- tional control plnys in muscle development it would be very useful to be able to titrate, directly, the celll~liir levels o f the myosiri RNA transcripts during ditTercntiation and development. The viral RNA- clcpcndcnt DNA polymerases. ;IS isolated from Kous s;ircom;i virus "41 iind avian myeloblilstosis virus 1 3 1 . have been used succcss~'ully in the synthesis of complementary DNAs (cIlNA) to various cellular nicssengers 126- 291. The cDNAs have been widely used ;is probcs in the Jelermination of the cellular Icvcls o f the spccitic tctiipLite sequences during various dcvclopmcntal processes ['h. 30.31 1. Recently we have used 26-S myosin mRNh. derived from the polysomes of 14-duy embryonic chick Icg muscle, as ;I tenipliitc l*or the synthesis of myosin complementary DNA 1321.

In this study 2 6 3 myosin mRNA dcrivcd from thc myosin mcsscngcr rihonuclcoprotein particles was utilized ;is ;I template I'or the gcneration of myosin cDNA. The probe was used to determine the concen- tration o f myosin sequences in cytoplasmic RNA iso- lated from tertiary cultures o f chick fibroblasts. RNA derived from primary cultures of myoblasts undergo- ing differentiation was also iinalyzed. From this data the number o f myosin informational transcripts pres- ent during the various stages of myogenesis was determined.

MATERIALS AND MtTHODS

~ ' o i i d i i i o t i . ~ Cell C'ultiiw

Pure cultures of tertiary chick fibroblasts were ;I generous gift of' thc Cniversity of Connecticut's Cell Culture Facility. They were obtained in the followinp manner: pathogen-free 10-day chick em- hryos were decapitated, minced and trypsinized. The initial cultures were seeded at approximately 2 x l o 7 celIs:lOO-mni culture dish. Thecells were grown in standard NCI medium supplemented with 6";, calf serum at 37 C in 5 " , , C 0 2 [43]. After 48 h the cells were collected. diluted with 2 vol. growth medium, ;wid reseeded at the initial density. Tertiary cultures were obtained in a similar manner. and allowed to grow to confluency before being harvested as outlined below.

Myoblast culiurcs were obtained from 1 I-day p:ithogcn-l'rec embryos. and grown ;is described pre-

viously [7.331. Only cells that excluded trypan bluc were counted in determining the number of cells to be plated.

Isoltilioii of' M p v i t i in R N A

26-S. poly(A)-containing RNA was isolated from the XU-- 1 2 0 3 region of a sucrose grudient derived from 12-day embryonic chick leg muscle as described previoudy [ 191. Under these conditions no detectable ribosomal RNA wiis found to bind to the olipo(ciT)- cellulose. The 96-S poly(A)-containing R N A was incubated in a wheat germ cell-free system iis pre- viously described (441. As shown in Table 1. 78",, of the rridioactivity incorporated into trichloroucetic- acid-precipitable material co-purified with ni\:osin after two steps of ionic precipitation and DEAE- cellulose chromiitography [23]. After sodium dodccyl- sultiiteiacrylamide gel electrophoresis, all the radio- activity eluting with myosin from the DEAE-cellulose column migrated with the 200000-M, myosin heuvy chain marker ils previously reported (441.

1.voliitioti ol R N A jsoi i i rhr Fihrohlust tit111 iW.Iddti.vt C1iltlrrl'.V

The procedure described below is essentiall\ the method devised by Morse ('I (11. [34] and Teppernlan r t (11. [33] with soiiie slight moditiciitions. All opcr.1- tions were performed at 4 C. After decanting the medium. the cultures were rapidly cooled by three rinses with ice-cold 0.02 M Tris-HCI (pH 7.4). 0.25 M NH4CI and 0.01 M MgC12 (buffer 1 ) . The cells were then scraped from the plate with a rubber policcman in a small volume of buffer 1 and pelleted by centri- I'ugation at I000 x g for S min. The cells were lysed in 5 x the packed cell volume of buffer 1 , containing 0.5 Triton X-100, by 20 passuges through a sniiill- bore pasteur pipet. The lysate was centrifuged at l2000xg for 10min at 4 C. and the supernatant layered on t o a 27-ml 15 - 40 ":, sucrose gradient made up in buffer 1. The sucrose gradients were centrifuged in an SB 110 rotor (IEC) at 90000xg for 90 min at 4 C. The absorbance of the gradient was monitored by a Gilford spectrophotometer equipped with ti flow cell. and all material sedimcnting faster than 60 S Wiis collected. This was diluted with an equiil volume of buffer I , and the monosomes and polysomes pelleted by centrifugation at 200000xg for 160 min at 4 C . The pellet was extracted three times with phenol ils described by Aviv and Leder [MI. The RNA was precipitated by the addition of 1,3 vol. of 1.0 M sodium acetate, pH 5.3. and 2.5 vol. of 95",, ethanol.

D N i l Dt*tcrniiitcrtioit titid Cdl Nutithcr

DNA determinations were carried out usinp thc iiiicrofluorimctric technique described by Kissane iind

I . Kcrbhins and S. M . tleywood 603

Robins [MI. In determining cell numbers, a value of 2.8 pg DNAInucleus was used [37].

I~c~tc~rniiiicitioii 4 ' r l i c . M,whlust Fibroblust Rutio t r i r t l Fu.~ioii Iii(1cJ.v

A 60-nim culture dish. plated at the same density ;IS the cultures used for the isolation of the RNA, was rinsed three times with 0.12 M NaCI, 5.6 niM KC'I and 2 mM CaC'I2, tixed for 1 - 2 h in methanol, and stained for 10 min with freshly filtered hema- toxylin (Fisher). The cells were then embedded in epoxy resin and the number of myoblasts present Jetermined by their morphology under microscopic examination. Such determinations arc subject to error in thut some niyoblitsts may be judgcd as fibroblasls. In this case our values would tcnd to be a minimum estimate of musclc cells present. The culture was also used to determine the fusion index, defined a s : (number nuclei in myotubes)i[nuniber of nuclei in (myot uhcs + myoblasts)].

. I Iccr .srr iv~ i~ ic~i i t of ' Rtrdiotrct ir i~~~~ Iiirwportrtccl into the, M,~) .v in I l e t i r ~ ~ * i'liuiii

The cclls were grown the indicated number or hours. 3 h prior to harvest, the medium was drained. :ind 3 in1 ol' I'rcsh mcdium containing 20 pCi of 3 mixture of IS -3H-labelcd amino acids (New England Nuclear-NEC 445) was added to the 100-mm plates. At the end of the 3-h pulse, the cells were harvested, pellcted and lysed as described above. An aliquot of the lysate was taken li)r ;I DNA determination \vhile 350 pg carricr niyosin was added to the rc- mainder of the sample, and the myosin precipitate was clcctrophorcscd on 5.5 sodium dodecylsulfate/poly- itcrylamide gels, which were then fixed and stained [38]. The 300000-M, myosin band was excised, and the radioactivity determined. The proportion of fibro- blasts in ;I parallcl culture was measured as outlined above, and a correction made. such that the expressed tigurcs represent the radioactivity incorporated into niyosini'mg myoblast-myotube DNA.

Srnthcsis o f 3 H- Luhc~l~~tl C'oni~~l~~i~~c~ntaryitur~~ D N A

Synthesis was carried out in a volume of 0.2 ml containing: 50 mM Tris-HC1 ( p H 7.9), 10 mM MgCIz, 60- 140mM KCI, 6mM dithiothreitol, 0.4mM dTTP, dCTP. dATP. and 0.15 mM of ['HIdGTP (specific activity 8.5- 9.4 Ciimmol). Approximately 2.0 pg niyosin niRNA was preincubated with an equimolar amount of oligo(dT),Z - (Collaborative Research) in 0.1 M NaCl at 37 'C for 10 min, and then added to thc reaction mixture along with 1.0-2.0 units of the RNA-dependent DNA polynierase from avian myelo- blastosis virus [25]. The reaction mixture was in-

cubated at 40 "C for 70 min, and the cDNA isolated as described previously [32].

tYuch~ic A cirl H j h r i d i x t ion

Hybridizations were carried out with approxi- mately 2500 counts/min cDNA in 5 pI containing: SO deionized formamide. 0.4 M NaCI. 0.05 M Hepcs (pH 6.8) 0.001 M EDTA. 0.1 'I.,, dodecylsulfate, and RNA as indicated in the figure legends. The samples were placed in glass capillaries. sealed. and incubated in a 95 C water bath for S min. Hybridization was carried out at 52 ' C to the indicated rot values. Duplex formation was assayed by S1 nuclease a s previously described [32]. Acid-precipitable radio- activity was collected on 0.45-pn Millipore filters (mixed ester type). In order to ensure quantitative recovery of the radioactivity present, the filters were treated according to a procedure developed by the New England Nuclear LSC Applications Laboratory. (LSC Applications Note I . New England Nuclear).

RESULTS

'The complementary DNA used in these studies has been described previously [32.40]. The cDNA was isolated (see Materials and Methods) and h \ - bridiired back to its template sequences. Xpprmi- matcly 50". of the radioactivity present with cDNA was Found to be resistant to S1 nuclease. The standard and double-reciprocal plots of the reaction kinctics are shown in Fig. 1 . We have found the double- reciprocal transform [39.41] to be useful in unambi- guously determining the rot! 2 of this type of reaction. rOt l / z is 2.0 f 1.4 M SKI. This value was used to deter- mine the fraction of myosin template sequences present in other samples of RNA by utilizing the relationship: fraction of myosin RNA = 2.0 f 1.4/rd1 z sample RNA.

The specificity of the cDNA as a probc for the myosin RNA sequences was tested by attempting to hybridize the cDNA to: E. coli rRNA, yeast tRNA. mouse globin mRNA (Searle Laboratories) and poly(A). Fig. 1 A demonstrates that none of the above showed any degree of hybrid formation with the cDNA. The lack of any hybridization with these heterologous RNAs confirms the stringency of the hybridization conditions. These results and the data presented in Table 1 suggest that the cDNA is pre- dominately a probe for myosin heavy chains. These results confirm the specificity of the probe and the stringency of the hybridization condition.

Myosin R N A Sequences in Tt)rtitirjg Cultuws of'Chick Fibroblasts

Fibroblasts synthesize small amounts of myosin [9]. The probe's ability to detect low levels of myosin

604 Quantification of Myosin Heavy-Chain mRNA during Myogenesis

A I c

c .L" 0 C m 0

m

- 0 0 3 7 D,

- I

c c

N

. - -1 0 1 2 3 0 0 25 0 50 0.75

log ro t /mot I-' s l / ro t ( rnol-' I s-' )

Fig. 1 . Stunrletrd und douhlc~-rc~ci~~r~iculplois o/ rhc kinc,tics of h)~hridi:crrion oJ rhr (,DNA p r q ) u r d / r o n i myosin m R R A protein. Iiybridizations were carried out as described in Materials and Methods. (A) (t -0) Template myosin mRNA; (W -.) E. coli rKNA; (A- -A) yeast tRNA; (0-- -0) poly(A): (A- -A) globin mRNA. (B) (0-0) Double-reciprocal plot of the hybridization back to the template RNA

Table 1. Ciipcrri/;it~urion ?/ rhu product o/2~-S-RNA-direc~tc~dprotcin . s j ~ ~ ~ / I ~ e ~ s i s in o wlieat gtwn cell,frce system with myosin heavy choin After incubation. 100 pg carrier myosin was added to each incuba- tion mixture. The purification by two successive precipitations at low ionic strength and DEAE-cellulose chromatography have been previously described 1231. All the radioactivity eluting with myosin from the I~EAl:-cellulose column migrated as a single peak at M, 200000 upon clectrophorcsi\ [44]

Control + 26-S Amount KNA copurifying

counlsjmin total

I c i t a l r ad iox t iv i ty 1272 9850 100 Ionic precipitation 1 365 7980 81 Ionic precipitation 2 320 7875 80 DEAE-cellulose 205 1656 I8

informational transcripts was tested by hybridizing the cDNA to total polysomal RNA obtained from 52-h, tertiary fibroblast cultures. The double-recipro- cal plot of the kinetics of hybridization is shown in Fig.'. The reaction is characterized by a rot1/2 of 1.075 x lo3, and the proportion of myosin RNA present in the sample in 0.00186. Other trials yielded slightly different values, the mean and standard de- viation of which are included in the data presented in Table 2.

In order to convert the above value into the number of myosin informational transcripts per cell, it is necessary to make the following two assumptions : the molecular weight of myosin mRNA is 2 x lo6 [lo] and the fibroblast nucleus contains, on the average, approximately 2.8 pg of DNA [37].

Then, the number of transcripts/nucleus is equal to:

Avogadro's number . -~ ~ - - x (yield of RNA) x 0.00186

2 x 10" - - - - ~ _ _ _ ~ _ _ - - .

yield of DNA 2.8 x 10-12

. -

- L

x n I .. -

I I I I J 01 0 2 4 6 8 , P, "

lo4. l / r o t (rnoi- ' I 5 " )

Fig. 2. Dolihlc~-rc~cil,roc.(IIplor of thc / c ~ ~ I ~ r i d i : ~ i t i ~ i t ~ of mj.o.\in (,L)R'A to jibroblast polrsomal RNA. RNA was isolated from 51-11. tertiary cultures of chick fibroblasts and hybridized to the myosin cDNA Tor 60 h. The rotl of this reaction is 1.075 x 10' mol I - ' s

A range of 500-850 transcripts/nucleus was ob- served. This figure, while being of intrinsic interest, also allows one to correct for fibroblast contamination in the myoblast cultures (Table 2).

Myosin m R N A in Myohlast Cultures during Development

The proportion of cytoplasmic myosin RNA sedimenting at greater than 80 S from the myoblast and myotube cultures during various stages of develop- ment was determined. RNA was isolated from 3.5, 24,48 and 67-h cultures and hybridized to the myosin cDNA. The double-reciprocal plots of the kinetics of hybridization, and the rOt l , 2 values of the reactions, are shown in Fig.3. From these data the fraction of myosin mRNA present in the overall cell population was calculated, and the number of transcripts per myoblast-myotube nucleus determined (Table 2 ) .

The fraction of myosin mRNA, present at dif- ferent stages of development, is shown in Fig.4. together with the fusion index of the cultures. As seen previously [6,10], fusion is essentially complete after

1 . Rohbins and S. M. Heywood 605

Pnr;imeter ~~~

Sample

fibroblasts 3.5 h -_ - -

24 h 48 h _ _ -

61 h (time after plating)

10 x cell density I nit iu I plating (cells t 00-m in pla tc) At harvest (cells 100-nim plate)

RNA DNA Fusion (",,) Fibroblast contamination ( "J Fraction of myosin RNA Mjosin RNA transcripts

26-S myosin RNA utilization myoblast-myotuhe nucleus"

(counts min-' ~ g - ' ) ~

20 -

0.53 -

-

0.0015 0.0004

500 - 850

20 10 0.15 0

23-21 0.0037 0.0005

400 - 500

0.25

12 13 0.37 0

21 -29 0.009 f 0.002

4 500

0.19

6 8.8 0.55

85-90 28 - 33 0.010 i 0.003

1 2 000

0.079

7 38 0.19

85 50-58 0.008 f 0.0017

2 200

1.2

Assuming a value of 600 myosin mRNA transcriptsifibroblast, the following correction was made to compensate for fibroblast conta- mination or the myogenic cultures:

Myosin RNA transcripts myoblast-myotuhe nucleus~

total number of myosin R N A transcripts - (600) (number of fibroblast nuclei) number of myoblast-myotuhe nuclei

. ~~~~ ~~ .~ - -

The DNA value and nuclei count are corrected for the percentage of fibroblast contamination (Fig.5). The ratio: (radioactivity in myosin heavy chain 'pg DNA),'(myosin heavy chain RNA transcripts/nucleus)

Fig. 3. Doirhl~-r.rc.i/)ro(.cil p10r.s o/' rlrc. Ir~~hricli:oiinn o/ nij.o.siii c D N A 10 poIJ.soniu1 RNA i.roluiivlJrorn i d 1 c-u//urr.s undrrgoiiig ni,i.ogtwc.ti.r. The polysomal RNA was isolated from the various cultures as described in Materials and Methods, and hybridized for 60 h with the myosin cDNA t o the indicatcd ii)/ values. The lincs drawn represent the lines of best fit, as determined from the least-squares linear regression of the data. ( A ) 3.5-h cultures: slope 8.6, roil 2 540 rnol I - ' s. (B) 24-h cultures; slope 6.3. r 0 i l ~ 2 178 mol I - ' s. (C) 48-h cultures; slope 4.1, roil 144 mol I - ' s. (D) 67-h cultures: slope 6.5. q,tl 2 235 mot I - ' s

48 h. Myogenesis is accompanied by a significant increase in the proportion of myosin informational transcripts present in the cytoplasmic RNA. However, the proportions may be misleading. Table 2 shows that both the degree of fibroblast contamination and RNA : DNA ratios fluctuate during myogenesis. A more useful indicator of the myosin RNA content of the myoblast-myotube population is the actual number o f myosin informational transcripts present per myo-

blast-myotube nucleus. The values were calculated (see Table 2) and the data plotted in Fig. 5, together with the radioactivity incorporated into the myosin heavy chain during a 3-h pulse. From Fig.5, and the data listed in Table 2, i t is obvious that the rate of synthesis of the myosin heavy chain (as measured by the radioactivity incorporated during the 3-h pulse) is not proportional to the level of myosin informa- tional transcripts present in the cytoplasm of the cells.

3 0!5

2 0010 n c

0 7.

F L - L

0 n. ? ' 0005

0001

DISCUSSION

The reassociation of the myosin cDNA with its template is characterized by a roil ,Z of2.0 M Ci. This relatively high valuc (when compared with other mRNA . cDNA reassociations) t26.271 indicates a highcr than expected nuclcotide complexity for the myosin niRNA template. This R N A preparation elcctrophoresed as a single band at 33 S on denaturing formaniidc polyacrylamide gels [lo]. The 26-S species was also shown to direct the synthesis of a single protein product when translated in the wheat germ cell-free system (Table 1) . This protein was charac- terized as the myosin heavy chain based on its ability to go through a three-step purification with carrier myosin and its subsequent co-migration with the carrier myosin on sodium dodecylsulfate,iacrylamide gels [44]. However, because 20",;, of the radioactivity incorporated into trichloroacetic-acid-precipitable ma- terial was lost during the purification of myosin (Table 1 ). a small amount of other poly(A)-containing RNA which directs the synthesis of other proteins may be present. This contamination would result in the higher than expected rotl '2. Chi et ul. have noted that there appears to be multiple developmental- stage-specific species of myosin [I 51. Therefore, our 2 6 4 inRNA preparation niay consist of a number of distinct myosin heavy chain messengers. If these various species were unable to cross-hybridize with

Tlme of cu l tu re ( h :

one another under our stringent reassociation con- ditions, the effective complexit> of the template prep- aration would be greatly increased. We also think i t likely that the combination o f high formamide con- centration and relatively high tempcrature of' incubn- tion also contributes to the higher than expected ridl 2 .

In confirmation of this hypothesis the reassociation of mouse 9-S globin mRNA (gift of J . B. Lingrel) was characterized by a roi l 2 of7.0-9.0 x M s - ' . a value substantially higher than those previously reported in the literature [21,42]. Thus, despite the relatively high rofl 2 value observed, we feel that the results suggest that the cDNA is predominentl) a probe for myosin heavy chain mRNA.

I t is important to bear in mind the source of the RNA used in the hybridizations. As noted in Materials and Methods, the various preparations of cell cultures were lysed with detergent, and the polysomes analyzed on sucrose gradients. The fractions collected contain mRNAs engaged in protein synthesis, the ribosomal RNA, and all messenger ribonuclear protein particles heavier than 80 S. Presumably the myosin ribonuclear protein particles, if present in these cells, would be included [19,23]. However, no effort was made in these studies to differentiate between the amounts of myosin RNA transcripts present in the stored RNA protein fraction and the myosin mRNA actively engaged in protein synthesis.

Although the process of detergent lysis is supposed- I ) ;I gentle one [17.33]. sonic nuclear leakage into the cytoplasm may occur. and the polysomes. cspecially i n the K N A . protein region. may be contaminated with nucleur RNAs. Howe\/er, of the 60000 counts: min incorporated into nuclear RNA after a 10-min pulse label ol' 42-11 muscle c d l cultures with ['HI- uricline o n l y I of the radioactivity is Ibund in the cytoplasmic fraction. therefore i t is unlikely that the degree 01' nuclear contamination is significant.

The ratio (radionctivity incorporated into myosin heavy chain,spg niyoblast-myotube DNA! number of transcripts~'niyoblast-myotube nucleus) provides an eslimate of the utilization of the 2 6 3 myosin KNA for the production of thc myosin heavy chain (Table2). Examination of these values show that the transla- tional modulation o f this KNA population during niyogenesis is quite pronounced. During the first 48 h 01' culture the trend is towards less efficient use of t hc myosin i n forrna t ionu I t rii nscripts. The myosin m R N A in the early myotubcs is the least efficient in potnoting the synthesi\ of' myosin. Thc trend. how- ever. is reversed by 67 11. such that. even though there are onl) I 5 ;IS man!. transci-ipts a s were prcscnt at 4X h . the rate of' myosin synthesis has increased by al i i iosr 3- l i ) ld (Fig. S),

I t seein\ reasonable to iihhitiiie that the earlier (34 mid 48-11) cultures contain: ( a ) a general pool of myosin mKNA that is. for some reason. lranslationally inefficicnt, or (b) a population ol'niyosin informational transcripts that is inactive. At this tinic i t is not clear which of these alternatives i \ thc major factor regulat- ing the utili7ation of myosin mRNA. However. studies using cell-frec systems suggest that both of these possibilities may play a role 117, 18.32).

Our results are in agreemcnt with a previous estimate of the number of myosin RNA transcripts i n post-fusion cultures. In ii ccll culture system derived I'rom quail embryonic pectoral muscle. Emerson and Beckner [ I I ] calculated that 2 -6 x lo3 myosin RNA transcripts nucleus arc needed in order to account for the rates of synthesis observed during the post- fusion burst of myosin synthesis. In our 67-11 cultures, when the rapid synthesis of niyosin is taking place. the hybridization data indicate that approximately 2200 RNA transcripts, myoblast-niyotube nucleus are present.

Using the cDNA it should now be possible ac- curatel), to determine the levels of myosin informa- tional transcripts present i n the various cellular com- partments during development. Determination of the temporal and spatial locutions of the inactive informa- tional transcripts and the kinetics of utilization should aid i n the elucidation of the controls underlying the synthesis of the myosin heavy chain.

I i i \ t i tuic.(;r : ini A11733 and NIH ( i r ~ i t HDO3310. Jellrcq l<ohhiii> I'lii\ ucirb w a s \clpportcd h) gi;inl\ I'roi11 The N;i~~c*naI C'anccr

608 J . Robbins and S. M. Heywood: Quantification of Myosin Heavy-Chain mRNA during Myogenesis

33. Tepperman. K.. Morris, G., Essien, F. & Heywood, S. M.

34. Morse. R.. Herrrnann, H. & Heywood, S. M. (1971) Biochim.

35. Aviv. H. & Lxder, P. (1972) Pror.. Nut1 A d . Sci. U.S.A. 69.

36. Kissane. J . M. & Borins, E. (1958) J . Biol. Chmi. 233,184- 188. 37. Davidson. J . N. & Leslie, I . (1950) C'unwr Rus. 10. 587-594. 38. Weber. K.&Osborn, M.(1969).1. Bid. C/iem.244,4406-4412. 40. Robbins. J . (1976) T/ic ftx,pururion und Charnclrrizurion of

A4.vo.h Coniplcnicw/urj~ DNA. Ph.D. Thesis, University of Connecticut. Storrs, Connecticut 06268.

(1975) J . C'<>Il. P / i j ~ . ~ i o l . 86, 561 - 565.

L ? ~ ( J ~ / I , I x . Ac,ta. 232, 403 -409.

140X- 1412.

41. Birnstiel, M. L., Sells, B. H . & Purdom, I . F. (1972) J . Mol.

42. Young, B. D., Harrison, P. R., Gilmour, R. S., Birnie. G. D.. Hell, A,, Humphries, S. & Paul, J . (1974) J . Mol . Biol. 84.

43. Marcus, P. I. & Carver, D. H. (1965) Scirncr I Wadi.. D.C.1

44. Kennedy, D. S. & Heywood, S. M. (1976) FEBS Lett. 72.

Bid . 63, 21 - 39.

555 - 568.

14Y, 983 - 986.

314-318.

J . Robbins and S. M. Heywood, Genetics and Cell Biology Section, University of Connecticut, Storrs, Connecticut, U.S.A. 06268