Developmentally regulated troponin C mRNAs of chicken skeletal muscle
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CRAIGBEREZOWSKY AND JNANANKUR BAG University of Guelph, Department of Molecular Biology and Genetics, Guelph, Ont., Canada N l G 2WI Received June 4, 1991 BEREZOWSKY C., and BAG, J. 1992. Developmentally regulated troponin C mRNAs of chicken skeletal muscle. Biochem. Cell Biol. 70: 156-165. Fast and slow/cardiac troponin C (TnC) are the two different isoforms of TnC. Expression of these isoforms is developmentally regulated in vertebrate skeletal muscle. Therefore, in our studies, the pattern of their expression was analyzed by determining the steady-state levels of both TnC mRNAs. It was also examined if mRNAs for both isoforms of TnC were efficiently translated during chicken skeletal muscle development. We have used different methods to determine the steady-state levels of TnC mRNAs. First, probes specific for the fast and slow TnC mRNAs were developed using a 390 base pair (bp) and a 255 bp long fragment, of the full-length chicken fast and slow TnC cDNA clones, respectively. Our analyses using RNA-blot technique showed that fast TnC mRNA was the predominant isoform in embryonic chicken skeletal muscle. Following hatching, a significant amount of slow TnC mRNA began to accumulate in the skeletal (pectoralis) muscle. At 43 weeks posthatching, the slow TnC mRNA was nearly as abundant as the fast isoform. Furthermore, a majority of both slow and fast TnC mRNAs was found to be translationally active. A second method allowed a more reliable measure of the relative abundance of slow and fast TnC mRNAs in chicken skeletal muscle. We used a common highly conserved 18-nucleotide-longsequence towards the 5 '-end of these mRNAs to perform primer extension analysis of both mRNAs in a single reaction. The result of these analyses confirmed the predominance of fast TnC mRNA in the embryonic skeletal muscle, while significant accumulation of slow TnC mRNA was observed in chicken breast (pectoralis) muscle following hatching. In addition to primer extension analysis, polymerase chain reaction was used to amplify the fast and slow TnC mRNAs from cardiac and skeletal muscle. Analysis of the amplified products demonstrated the presence of significant amounts of slow TnC mRNA in the adult skeletal muscle. Key words: troponin C mRNA, skeletal muscle, cardiac muscle, polymerase chain reaction, developmental changes in muscle.
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BEREZOWSKY C., et BAG, J. 1992. Developmentally regulated troponin C mRNAs of chicken skeletal muscle. Biochem. Cell Biol. 70 : 156-165. La troponine C (TnC) rapide et la troponine C lente/cardiaque sont deux isoformes difftrentes. L'expression de ces isoformes dans les muscles squelettiques des verttbrts est soumise a une rkgulation dtveloppementale. Ainsi, nos etudes concernant leur expression ont porte sur les taux des d e w types de mRNA A I'ttat d'tquilibre. Nous avons Cgalement Cvalut I'efficacite de la traduction des mRNA des deux isoformes de la TnC au cours du dheloppement du muscle squelettique chez le poulet. Nous avons utilist diverses mtthodes afin de dtterminer les taux des mRNA a l'ttat d'tquilibre. Utilisant des fragments longs de 390 paires de bases (pb) et de 255 pb, obtenus respectivement de clones pleine longueur des cDNA de la forme rapide et de la forme lente de la TnC de poulet, nous avons d'abord dtveloppt des sondes sptcifiques des mRNA de la forme rapide et de la forme lente. La technique de buvardage du RNA a permis de montrer que le mRNA de la forme rapide de la TnC est l'isoforme prtdominante dans le muscle squelettique embryonnaire du poulet. Suite a l'tclosion, une quantitt importante du mRNA de la forme lente de la TnC s'accumule dans le muscle squelettique (pectoral). Quarante-trois semaines aprts llCclosion, le mRNA de la forme lente de la TnC est presqu'aussi abondant que celui de l'isoforme rapide. De plus, la majoritt des mRNA de la forme lente ainsi que de la forme rapide montrent une activite traductionnelle. Une seconde mtthode a permis une mesure plus fiable de la quantitt relative des mRNA de la forme lente et de la forme rapide de la TnC dans le muscle squelettique de poulet. Nous avons utilist une stquence commune, trts conservte, longue de 18 nucltotides et localiste vers la terminaison 5 ' de ces mRNA afin d'effectuer une analyse par extension d'amorce de ces d e w mRNA au cours d'une seule rtaction. Les rtsultats de ces analyses confirment la prtdominance du mRNA de la forme rapide de la TnC dans le muscle squelettique embryonnaire tandis qu'une accumulation importante du mRNA de la forme lente de la TnC est observte dans le muscle squelettique (pectoral) du poulet suite a I'klosion. En plus de l'analyse par extension d'amorce, l'amplification tlective (polymerase chain reaction) fut utilist pour amplifier les mRNA de la forme rapide et de la forme lente de la TnC du muscle squelettique ainsi que du muscle cardiaque. L'analyse des produits amplifits a dtmontrt la prtsence de quantitts importantes de mRNA de la forme lente de la TnC dans le muscle squelettique du poulet adulte. Mots elks : mRNA de la troponine C, muscle squelettique, muscle cardiaque, polymerase chain reaction, changements dCveloppementaux dans le muscle. [Traduit par la rtdaction]
Introduction The contractile proteins of skeletal and cardiac muscles exist in polymorphic forms, which are characteristic of the muscle fiber type. Gene expression of each member of an isoprotein family is regulated i n a tissue specific a n d developmental stage specific manner (Buckingham a n d Minty 1983). To understand how the switching o f synthesis f r o m one member of an isoprotein family t o the other occurs, we have chosen to examine this process during development o f chicken skeletal muscle using T n C as o u r model. T n C is one of the three troponin subunits that form a functional complex which regulates ABBREVIATIONS: TnC, TnT, and TnI, troponins C, T, and I; bp, base pair@);NP-40, Nonidet P-40; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; HBSS, Hanks' balanced salt solution; DMEM, Dulbeco's modified Eagle's medium; arcC; cytosine arabinoside; BSA, bovine serum albumin; BRL, Bethesda Research Labs; M-MLV, Moloney murine leukemia virus; PCR, polymerase chain reaction; Taq, Thermus aquaticus; a-Tm, a-tropomyosin. Printed in Canada / Imprime au Canada
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BEREZOWSKY AND BAG
the calcium-mediated contraction of striated muscle. While TnT and TnI have many known isoforms including fast, slow, and cardiac, TnC has only two known isoforms: fast and slow/cardiac (Buckingham and Minty 1983; Ebashi and Endo 1968; Gahlmann and Kedes 1990; Mannherz and Goody 1976). The slow TnC of embryonic skeletal muscle and the TnC of cardiac muscle have identical amino acid sequences and are considered to be the product of the same gene (Wilkinson 1980), whereas the fast TnC found only in adult skeletal muscle is the product of a different gene (Maisonpierre et al. 1987; Wilkinson 1980). In studies using monospecific antibodies to detect fast and slow TnC polypeptides, it was reported that during skeletal muscle development the slow or cardiac gene undergoes isogene switching and its expression is repressed in adult skeletal muscle (Toyota and Shimada 1981, 1983). In cardiac muscle, however, the cardiac TnC gene is expressed in embryonic as well as in adult heart (Toyota and Shimada 1981, 1983). The same gene (cardiac/slow) is thus regulated by different developmental programs in skeletal and cardiac muscle. The significance of developmental regulation of muscle proteins is not clear. It may be that the slow isoform of TnC is specific for sarcomere assembly during development of both skeletal and cardiac muscle in embryos and is replaced by the appropriate isoform in adult skeletal muscle. A single TnC isoform, however, appears to be sufficient during cardiac muscle development. It is probable that a large number of isoforms for contractile proteins may best fulfill the precise structural and kinetic requirements of each muscle fiber type at different stages of development. This implies that isogene switching may also be controlled by exercise and nerve influence (Swynghedauw 1986). To examine the changes in the expression of fast and slow TnC mRNAs during muscle development, the steady-state levels of these were measured. Furthermore, we have previously reported the presence of significant amounts of slow TnC mRNA in adult skeletal muscle (Berezowsky and Bag 1988) when previous studies using TnC antibody did not detect any slow TnC polypeptide in this muscle (Toyota and Shimada 1981, 1983). Therefore, have examined the subcellular distribution of TnC mRNA to determine if a significant amount of slow TnC mRNA is repressed from translation at any stage of muscle development in ovo or in cultured muscle cells. Materials and methods Subcellular fractionation and isolation of RNA The total cellular RNA from chicken skeletal and cardiac tissues was isolated using a modification of the guanidine - hot phenol procedure as previously described (Maniatis et al. 1982). To isolate polysomal and postpolysomal RNAs, the appropriate subcellular fractions were first isolated by a modification of the procedure as previously described by Bag and Sarkar (1975). Briefly, 1 g of the excised tissue was ground to a fine powder in liquid nitrogen and approximately 5 mL of lysis buffer (25 mM Tris-HC1 (pH 7.5), 250 mM NaCl, 5 mM MgCI,, 0.5% NP-40, 5 mM DTT, 50 pg cycloheximide/mL, 200 pg heparin/mL was added to the tissue. This mixture was homogenized in a dounce homogenizer with 20-30 strokes and examined under a microscope to ensure complete cell lysis. This mixture was then centrifuged at 8000 x g for 15 min at 1°C, and the postnuclear supernatant containing the polysomes was collected and adjusted to 500 pg heparin/mL. To pellet and isolate the polysomes, this postnuclear supernatant was centrifuged at 100 000 x g for 1 h at 1°C. The postpolysomal
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supernatant was adjusted to 0.5% SDS and heated at 65°C for 5 min prior to RNA isolation. The polysomal pellet was dissolved in 10 mM Tris-HCI (pH 8.0) - 200 mM NaCl - 0.5% SDS. RNA was isolated from the polysomal and postpolysomal fractions by standard phenol-chloroform extractions as previously described (Maniatis et al. 1982). The final aqueous phase was adjusted to 0.2 M sodium acetate (pH 5.2) and the RNA was precipitated with 2.5 volumes of 100% ethanol at - 20°C. The RNA was pelleted at 8000 x g for 30 min, washed twice with 66% ethanol, dried briefly under vacuum, and dissolved in distilled H20. The concentrations of RNA in these samples were determined by measuring the absorbance at 260 nm and the integrity of the RNA in these preparations was determined by staining with ethidium bromide following agarose gel electrophoresis. The RNA samples were stored at - 80°C. Isolation of subcellular fractions from cells in culture Before harvesting, the cells were incubated with 20 pM emetine for 10 min at 37OC to prevent dissociation of polyribosomes during subcellular fractionation. The cells were washed four times (5 m ~ / 1 0 0 - c mplate) ~ with washing solution (130 mM NaCl, 5 mM KCI, 7.5 mM M MgCl,, 50 pg cyclohexamide/mL) and the cells were lysed on the plate by adding 4.0 mL of lysis solution (25 mM Tris-HC1 (pH 7.5), 250 mM NaCI, 5 mM MgCl,, 5 mM DTT, 0.5% NP-40, 50 pg cycIohexamide/mL, 200 pg heparin/mL. The cell lysate was prepared by homogenizing in a dounce homogenizer with 20-30 strokes. The cells were checked for complete lysis by examining the presence of nuclei free of cytoplasmic tags using a phase-contrast microscope. Cell culture Primary cultures of cardiac myocytes were prepared from cardiac tissue as previously described (Claycomb 1979; Malhotra and Bag 1987). Briefly, the cardiac tissue of 14-day-old embryonic chicken was treated with 0.1% collagenase and 0.1 Yo hyaluronidase for 15 min at 37OC. Multiple digestions were carried out. The cells obtained from the first two digestions were mostly fibroblasts and were discarded. Cells released by subsequent digestions (3-10) were pooled and the cardiac myocytes were separated from the fibroblasts by pelleting at 200 rpm (4 x g). Cardiac myocytes were plated at a desity of 2 x lo5 cells/cm2 and maintained in CMRL 1066 medium containing glutamine, 10% horse serum, 3% fetal bovine serum, and 40 pg insulin/mL (25 IU/mg, Sigma). The medium was supplementedwith 0.1 rnM 5 ' -bromo-2'-deoxyuridine during the first 24 h of incubation to select against dividing fibroblasts. Primary cultures of skeletal muscle cells were isolated from 12-day-old chicken embryos. In short, the skeletal muscle isolated from the breast and legs was minced into fine pieces with a scalpel. The minced tissue was placed in a sterile tube and washed twice with HBSS (Gibco) on ice. The tissue was then digested with 0.1% trypsin (Gibco) in HBSS at 37°C with agitation for 10-15 min. The digested tissue was filtered through a nylon membrane to remove any undigested material and this filtrate was then centrifuged at 1000 x g for 5 min at 4°C. The supernatant was removed and the pellet of skeletal muscle cells was suspended in DMEM containing 5% horse serum and 2% chick embryo extract (Gibco). The cells were plated on gelatin-coated plates at a cell density of 2 x 10' cells/cm2. The nutrient medium was supplemented with araC (Sigma) 24-48 h after plating to remove the dividing fibroblast cells. Gel electrophoresis of RNA and Northern blot analysis RNA was denatured in the presence of 50% formamide and 2.2 M formaldehyde before electrophoresis in 1% agarose gel containing 1.1 M formaldehyde and 10 mM sodium phosphate as previously described (Meinkoth and Wahl 1984). RNA from the agarose gel was transferred to a Zeta-probe blotting membrane (Bio-Rad) using 20 x SSC (1 x SSC: 0.15 M NaCl - 15 mM sodium citrate, pH 7.0). Following transfer, the membrane was baked at 80°C for 2 h and then washed in 0.1 x SSC - 0.5% SDS
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FAST TROPONIN C
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EcoRI
HinfI
HinfI
EcoRI
SLOW TROPONIN C
Hind111
Sau3AI
SalI
FIG. 1. Partial restriction map of slow and fast TnC cDNAs. Full-length fast and slow TnC cDNAs (Putkey et al. 1987; Reinach and Karlsson 1988) were subcloned into the multicloning site of vector pSPT18 (Pharmacia). Selected restriction sites are indicated by arrows. The 390-bp fast TnC HinfI and 255-bp slow TnC SadAI-SalI fragments are shown by hatched boxes. at 65°C for 30-60 min. Prehybridization was for 16-24 h at 65°C in 1% BSA (fraction 5) - 1 mM EDTA - 0.5 M NaH2P04 (pH 7.2) - 7% SDS. Hybridization was performed in the same buffer. A nick translation kit (IBI, Canada) was used to label probes with [ C Y - ~ ~ P ] ~ C (ICN; T P 3000 Ci/mM, 1 Ci = 37 GBq) and hybridization was performed for 48 h at 65°C. The membranes were washed two times for 30 min in 0.5% BSA (fraction 5) 1 mM EDTA - 40 mM NaH2P04 (pH 7.2) - 5% SDS at room temperature, followed by two washings at 65°C in 40 mM NaH2P04 (pH 7.2) - 1 mM EDTA - 1% SDS for 30 min. The washed filters were exposed to Kodak X-AR film using Cronex intensifying screens for 48-72 h at - 80°C.
200 U M-MLV reverse transcriptase (BRL) at 42°C for 2 h, with addition of another 200 U M-MLV reverse transcriptase after 1 h of incubation. The reaction mixture was adjusted to 0.2 M sodium acetate (pH 5.2) and nucleic acids were precipitated with 2.5 volumes of 95% ethanol using 5 pg tRNA (wheat germ tRNA, Sigma) as the carrier. The primer extension reaction products were then analyzed by electrophoresison a 10% polyacrylamide sequencing gel containing 7.7 M urea (Maxam and Gilbert 1980). The gel was fixed in 10% acetic acid (v/v) - 10% methanol (v/v) for 15 rnin and rinsed with distilled H 2 0 before drying. The dried gels were exposed to Kodak X-AR film using Cronex intensifying screens for 24-48 h at - 80°C.
Slot blotting of DNA and primer extension analysis of RNA Slot blotting of DNA was performed on a Minifold I1 slot-blot apparatus (Schleicher and Schuell) according to the manufacturers directions. Briefly, the DNA sample was suspended in 400 pL of 10 mM Tris-HC1 (pH 7.0) - 1 mM EDTA. The sample was adjusted to 0.3 N NaOH and incubated at 65°C for 30 min before neutralizing with an equal volume of 2 M ammonium acetate (pH 7.0). The sample was immobilized under vacuum onto a Zetaprobe membrane (Bio-Rad) prewetted in 1 M ammonium acetate (pH 7.0). The membrane was processed for prehybridization and hybridization as described for Northern blotting of RNA. Primer extension analysis of RNA samples was performed by a modification of the method previously described (Maisonpierre et al. 1987). The primer employed was an 18-base oligomer (5'CCCAGCATCCTCATCACC3'),which is antisense to a sequence common to both fast and slow TnC mRNAs (Putkey et al. 1987; Reinach and Karlsson 1988). This primer would hybridize towards the 5 '-end of both messages (see Fig. 6). The 18 oligomer was 5'-end-labelled using [Y-~~PIATP (ICN; 7000 Ci/mM) and polynucleotide kinase (BRL) (Maniatis et al. 1982). The labelled oligomer was purified from unincorporated radioisotope by gel filtration using a Bio-Gel P-4 column (Bio-Rad). The purified oligomer (50 ng) was added to the RNA sample (5 pg) in buffer containing 5 mM NaH2P04 (pH 7.2) and 5 mM EDTA, in a volume of 10 pL. The sample was denatured at 85°C for 6 min and annealed on ice for 10 min. The nucleic acid from the reaction mixture was precipitated and brought to a final volume of 20 pL in a buffer containing 50 mM Tris-HC1 (pH 8.3 at 42"C), 8 mM MgCI,, 30 mM KCl, 1 mM DTT, and 2 mM each of four dNTPs. Extension of the radiolabelled primer was carried out using
Synthesis of cDNA and its amplifcation by PCR Reverse transcription of both fast and slow TnC mRNAs was performed with the common antisense oligomer (Fig. 6) as the primer. One microgram of total cellular RNA was mixed with 100 ng of primer and heated for 6 min at 85°C. The reaction mixture was then cooled to 50°C and annealed for 10 min. Reverse transcription was performed as previously described (Sambrook et al. 1989) with 200 U MMLV reverse transcriptase (BRL) at 50°C for 30 min. Synthesis of double-stranded cDNA and subsequent amplification of the product was done with 100 ng of an oligomer primer specific for fast or slow TnC mRNA (Fig. 9) and 1 U Taq DNA polymerase (BRL) as previously described (Sambrook et al. 1989). Amplification was performed for 25-30 cycles in a Perkin Elmer Cetus thermocycler (1 cycle: 70 s at 94"C, 120 s at 5S°C, and 180 s at 72°C). The PCR amplification product was analyzed by electrophoresis in nondenaturing 8% acrylamide gel. The expected size DNA was electroeluted from the gel and further characterized by digestion with appropriate restriction enzymes. The electroeluted DNA was cloned into pBluescript I1 KS + / (Stratagene) after treatment with the Klenow fragment of DNA polymerase I and polynucleotide kinase (BRL). The DNA insert of the clone was sequenced using the dideoxychain termination method of Sanger (Sanger et al. 1977) employing a Sequenase V2.0 kit (United States Biochemical Corporation).
Results Subcellular distribution of TnC and tropomyosin mRNAs Results of a number of earlier studies (Mikawa et al. 1981; Toyota and Shimada 1981) have shown that the embryonic
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BEREZOWSKY AND BAG
FIG. 2. Analysis of the specificity of TnC cDNA probes. Purified full-length cDNA inserts of both fast and slow TnC cDNA clones were blotted onto Zeta-probe (Bio-Rad) nylon membrane as described in Materials and methods. Fast and slow specific probes (see Fig. 1) were nick-translated and hybridized as described under Materials and methods. (i) Probed with the 390-bp HinfI fragment of fast TnC cDNA; (ii) probed with the 255-bp Sau3AISan fragment of slow TnC cDNA. Full-length fast (Sk) and slow (Cd) TnC cDNA inserts were immobilized for hybridization: (a) 2.5 ng; (b) 5.0 ng DNA. The arrows indicate the positions of 2.5 ng of pBR322 DNA. skeletal muscle of chicken consists of both fast and the slow isoforms of the TnC polypeptide, whereas the adult chicken skeletal muscle is composed entirely of fast TnC. Previous studies from our laboratory, however, have shown that a significant amount of slow TnC mRNA is present in the adult chicken skeletal muscle (Berezowsky and Bag 1988). It is possible that the discrepancy between these results could be due to failure of translation of slow TnC mRNA in adult skeletal muscle. We have, therefore, first examined if the TnC mRNAs are efficiently translated at different stages of development of skeletal muscle. This was analyzed by examining the distribution of TnC mRNAs between the translationally active polysomal and inactive postpolysomal fractions of skeletal breast (pectoralis) muscles. The nonhomologous quail slow TnC cDNA clone used in our previous studies (Berezowsky and Bag 1988), however, hybridized to both fast and slow TnC mRNAs (results not shown). Therefore an attempt was made to obtain probes specific for fast and slow TnC mRNAs. The full-length chicken slow and fast TnC cDNAs (Putkey et al. 1987; Reinach and Karlsson 1988) were also found unsuitable for this purpose. Particularly the full-length slow TnC cDNA showed some cross-hybridization to the fast TnC mRNA (results not shown). Analysis of the homology of nucleotide sequences between different regions of the fast and slow TnC mRNAs using the DNAsis sequence comparison program (Hitachi Biologicals) suggested that the 390-bp-long HinfIdigested restriction fragment of the 5'-end of fast TnC cDNA clone and the 255-bp-long Sau3AI- and San-digested restriction fragment of the 3 '-end of slow TnC cDNA clone (Fig. 1) could be used as fast and slow specific probes, respectively. The fast probe exhibited a maximum of 38% homology within some region of the slow TnC mRNA. On the other hand, the slow specific fragment showed a maximum of 50% homology within some region of the fast TnC mRNA. T o determine if hybridization under stringent conditions would allow detection of a specific TnC mRNA using these fragments as probes, hybridization of nick-translated probes with full-length inserts of both fast and slow TnC cDNAs
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FIG. 3. Northern blot analysis of fast and slow TnC mRNAs of skeletal and cardiac muscles. Poly(A)-rich RNA from skeletal and cardiac muscles were isolated from the total cellular RNA samples using oligo(dT)-cellulose chromatography as previously described (Maniatis et al. 1982). One microgram of each poly(A)rich RNA was electrophoresed in an agarose gel and used for RNA blotting. Skeletal (Sk) and cardiac (Cd) muscle poly(A)-rich RNAs were probed with either the 390-bp fast TnC fragment (Sk' and Cd') or with the 255-bp slow TnC fragment (Sk and Cd). was examined by slot-blot analysis. The result presented in Fig. 2 shows that no detectable cross-hybridization of these probes was observed. Furthermore, Northern blot analyses of skeletal and cardiac muscle mRNAs (Fig. 3) show that the fast TnC cDNA fragment did not hybridize to the TnC mRNA of cardiac muscle. In contrast, the slow TnC cDNA fragment produced a strong signal when RNA from either skeletal or cardiac muscle of a 40-week-old bird was used (Fig. 3). Since there was no cross-hybridization between the slow 255-bp fragment and the full-length insert of fast TnC cDNA in the slot-blot analysis, it was concluded that the hybridization of the slow TnC cDNA probe with the skeletal muscle RNA was due to the presence of significant level of slow TnC mRNA in skeletal muscle. These studies show that the 390- and 255-bp restriction fragments of fast and slow TnC cDNA clones can be used to determined the steadystate levels of fast and slow TnC mRNAs, respectively. Therefore, we have used these fragments to measure the levels of fast and slow TnC mRNAs in the polysomal and postpolysomal fractions of skeletal muscle at various stages of development. The result of Northern blot analysis of skeletal RNA samples probed with the fast and slow TnC mRNA specific probes is shown in Fig. 4. Nearly all of the fast TnC mRNA of 14-day-old embryonic skeletal muscle was in the polysome fraction (Fig. 4, panel i). However, in the 21- and 43-weekold birds, a large proportion (21-26%) of the fast TnC mRNA was found in the postpolysomal fraction. When the same RNA samples were probed with the slow TnC cDNA fragment (Fig. 4, panel ii), no detectable level of the slow TnC mRNA was found in the breast muscle of 14-day embryo. At 21 weeks posthatching, detectable levels of the slow TnC mRNA were found in both polysomal and post-
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63
34
I4
A
a
B
b
C
c
62
17
53
19
D
d
E
e
FIG. 4. Subcellular distribution of mRNAs in chicken skeletal muscle. Northern blot analysis of chicken skeletal muscle RNA was performed as described in Materials and methods. Fifteen micrograms of the polysomal RNA (upper case letters) and an equivalent amount of the postpolysomal (lower case letters) RNA were used for analysis. (i) RNA samples were probed with the 390-bp fast TnC cDNA fragment; (ii) RNA samples were probed with the slow 255-bp TnC cDNA fragment. (A, a) Fourteen day embryo; (B, b) hatchling; (C, c) 2 weeks posthatching; (D, d ) 21 weeks posthatching; (E, e) 43 weeks adult. (iii) RNA samples were probed with chicken a-Tm cDNA. (A, a) Fourteen day embryo; (B, b) hatching; (C, c) 2 weeks posthatching; (D, d ) 43 weeks posthatching. The autoradiograms were scanned and the peak area was measured. The numbers above each lane represent the area corresponding to the signal in an arbitrary unit.
polysomal fractions (Fig. 4, panel i, lanes D and d). In 21and 43-week-old birds, 22-35% of slow TnC mRNA was also found in the translationally repressed postpolysomal fraction. Since there was no detectable hybridization of the slow TnC probe to the RNA samples from the breast muscle of 14-day embryo and hatchling, it is unlikely that the signals detected in the breast muscle of older birds was due to crosshybridization of the probe with the fast TnC mRNA. These results (Fig. 4 and Table 1) therefore show that detectable levels of slow TnC mRNA is present in the adult skeletal muscle. Furthermore, no significant difference in the distribution of fast and slow TnC mRNAs between the translationally active polysomal and repressed postpolysomal fraction was observed. The major portion (64-78s) of both TnC mRNAs was found in the polysomal population.
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Therefore, preferential repression of slow TnC mRNA translation was not detected. Our results presented here show that in the adult chicken breast muscle approximately 40% of total TnC mRNA consists of the slow isoform. In contrast, the fast TnC mRNA was the only TnC isoform detectable in the embryonic skeletal muscle. Accumulation of the slow TnC mRNA began following hatching. Our results also suggest that in contrast to the embryonic skeletal muscle where TnC mRNA was not detectable in the translationally repressed postpolysomal fraction, 22-36% of cytoplasmic TnC mRNAs of adult skeletal muscle were found in this fraction. To examine if this was specific for TnC mRNAs, we analyzed the subcellular distribution of mRNA for another contractile protein of the thin filament. The result of these analyses using an a-Tm specific probe (MacLeod 1981) is shown in panel iii of Fig. 4. The a-Tm mRNA was found to be translated efficiently in the skeletal muscle of up to the 2-week-old chicken. In adult birds, however, approximately 36% of a-Tm mRNA was also found in the translationally repressed postpolysomal fraction (Fig. 4 and Table 1). This pattern of subcellular distribution of TnC mRNAs and a-Tm mRNA was reproducible in several experiments using different RNA samples. Approximately 10% difference in the subcellular distribution profile was noticed between experiments. In adult birds, the presence of a substantial portion of the mRNAs in the repressed fraction may be due to their general inability to translate mRNAs efficiently. Also, the possibility of dissociation of polyribosomes during the isolation procedures cannot be completely ruled out. All the procedures, however, for subcellular fractionation included cycloheximide to prevent dissociation of polyribosomes. Developmental changes in the distribution of TnC mRNAs between polysomal and postpolysomal fractions in whole tissues prompted us to study whether similar changes occurred in cardiac and skeletal muscle cells in culture. In these analyses we used the quail cDNA probe because it hybridizes efficiently to both fast and slow TnC mRNAs. The result of the steady-state levels of polysomal and postpolysomal cytoplasmic TnC mRNA(s) of skeletal and cardiac muscle cells at various times after plating is shown in Fig. 5. The results show that in cardiac muscle cells, there was no significant change in the steady-state level of TnC mRNA between 4 and 14 days after plating of cardiac myocytes. Furthermore, TnC mRNA was not detected in the postpolysomal fraction (Fig. 5, panel i). In skeletal myotubes (Fig. 5, panel ii) the level of TnC mRNA was maximum at 14 days after plating, and like the cardiac myocytes, no TnC mRNA was detectable in the repressed fraction. It appears that TnC mRNA was translated efficiently in both cardiac and skeletal muscle cells in culture during the period of sampling in our studies. Therefore, translation of TnC mRNA in these cells in culture resembled embryonic and early neonatal tissues in chicken. In examining the relative steady-state levels of fast and slow TnC mRNAs during development of skeletal muscle using Northern blot analysis, one problem is variation in the specific activity between probes. Since both fast and slow messages migrate in the same region of 1-1.5% agarose gels and thus are not resolveable as two distinct bands, separate Northern blots need to be processed to determine the levels of fast and slow TnC mRNA for each sample. In addition
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TABLE1 . Subcellular distribution of mRNAs at various stages of skeletal muscle development Distribution of mRNA (%)
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Slow TnC
Fast TnC
Develo~mentstage
Polysomal
Free'
Polysomal
14-day embryo Hatching 2-week posthatching 21-week posthatching 43-week posthatching
-
-
100 100 100 78 74
100 64 78
-
36 22
Freea
Polysomal
-
100 100 90 ND 64
NOTE:These values were obtained by scanning the RNA blot shown in Fig. 4. ND, experiments not performed. 'Free refers to the postpolysomal fraction.
the possibility of hybridization of slow TnC probe with the fast TnC mRNA and vice versa cannot be completely ruled out in RNA-blot analysis. To circumvent these problems we used primer extension analysis using a common primer to determine the steady-state levels of both fast and slow TnC mRNAs in a single reaction. The nucleotide sequences of fast and slow TnC mRNAs were compared to find a common oligonucleotide sequence that could be used for primer extension of both mRNAs. A highly conserved 18-nucleotide-long sequence close to the 5 '-end and common to both fast and slow TnC mRNAs of chicken, mouse, and human has been detected (Parmacek and Leiden 1989; Putkey et al. 1987; Reinach and Karlsson 1988). This nucleotide sequence is shown in Fig. 6. These conserved 18 nucleotides are present between nucleotide 148 and 165 of slow TnC mRNA and between nucleotide 177 and 194 of fast TnC mRNA. Therefore, an antisense oligonucleotide to this conserved region would hybridize to both fast and slow TnC mRNAs and could produce primer extended products of 194 and 165 nucleotides in length, respectively. This possibly would allow us to determine the relative levels of both mRNAs using a single reaction. If a 5 '-end-labelled oligomer is used as a primer, the extended products could be quantified by using autoradiography. We initially examined if conditions employed for primer extension was within the linear range of response to the concentration of mRNAs. The result of these analyses is shown in Fig. 7. Increasing concentrations of poly(A)+ mRNAs from breast and cardiac muscle of 14-day-old chick embryos were used for primer extension. The results show that breast muscle mRNAs produced a 194 nucleotide long major primer extended product (lanes A'-C'). On the other hand, cardiac mRNA produced one major band (lanes A-C) and a distinct minor band. As judged from the molecular size markers (not shown), this major band appeared to be larger than 165 nucleotides in length. To determine the correct primer extended products of both fast and slow TnC mRNAs, the two bands shown by arrows (Fig. 7) were sequenced using the method described by Maxam and Gilbert (1980). Analysis of the nucleotide sequence confirmed that the 194-nucleotide-long primer extended product was derived from the fast TnC mRNA, and the band with an apparent molecular size of 185 nucleotides produced by primer extension of cardiac mRNA was in fact the correct product derived from the cardiac/slow TnC mRNA. Furthermore, the lower
aTIt,
-
22 26
Freea
10 ND 36
-, no detectable signal was found;
FIG. 5. Subcellular distribution of TnC mRNA in cardiac (i) and skeletal (ii) muscle cells in tissue culture. Northern blot analysis of 15 pg polysomal (upper case letters) and an equivalent amount of postpolysomal (lower case letters) RNA from cardiac myocytes (A-C, a-c) and skeletal myotubes (Af-C', a'-c'). RNA samples shown in panels i and ii were probed with quail TnC cDNA. (A, a, A ' , a') Four days; (B, b, B ' , b') 7 days; (C, c, C ' , c') 14 days after plating. Each of the lanes was scanned and the area under each peak was determined. The value corresponding to the signal in each lane is presented above the lane using an arbitrary unit.
molecular weight band corresponding to the cardiac TnC mRNA, found in the primer extension product of breast muscle mRNA, was also sequenced and found to be derived from the cardiac/slow TnC mRNA. It was concluded, therefore, that the slower migration of the cardiac/slow TnC mRNA derived primer extension product relative to the size
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BIOCHEM. CELL BIOL. VOL. 70, 1992
SLOW TROPONIN C 20 148 165 655 . . . ATG .../ / ...ggggaaGGTGATGAGGATGCTGGGgcagaa .A [ 1651
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CRAIGBEREZOWSKY AND JNANANKUR BAG University of Guelph, Department of Molecular Biology and Genetics, Guelph, Ont., Canada N l G 2WI Received June 4, 1991 BEREZOWSKY C., and BAG, J. 1992. Developmentally regulated troponin C mRNAs of chicken skeletal muscle. Biochem. Cell Biol. 70: 156-165. Fast and slow/cardiac troponin C (TnC) are the two different isoforms of TnC. Expression of these isoforms is developmentally regulated in vertebrate skeletal muscle. Therefore, in our studies, the pattern of their expression was analyzed by determining the steady-state levels of both TnC mRNAs. It was also examined if mRNAs for both isoforms of TnC were efficiently translated during chicken skeletal muscle development. We have used different methods to determine the steady-state levels of TnC mRNAs. First, probes specific for the fast and slow TnC mRNAs were developed using a 390 base pair (bp) and a 255 bp long fragment, of the full-length chicken fast and slow TnC cDNA clones, respectively. Our analyses using RNA-blot technique showed that fast TnC mRNA was the predominant isoform in embryonic chicken skeletal muscle. Following hatching, a significant amount of slow TnC mRNA began to accumulate in the skeletal (pectoralis) muscle. At 43 weeks posthatching, the slow TnC mRNA was nearly as abundant as the fast isoform. Furthermore, a majority of both slow and fast TnC mRNAs was found to be translationally active. A second method allowed a more reliable measure of the relative abundance of slow and fast TnC mRNAs in chicken skeletal muscle. We used a common highly conserved 18-nucleotide-longsequence towards the 5 '-end of these mRNAs to perform primer extension analysis of both mRNAs in a single reaction. The result of these analyses confirmed the predominance of fast TnC mRNA in the embryonic skeletal muscle, while significant accumulation of slow TnC mRNA was observed in chicken breast (pectoralis) muscle following hatching. In addition to primer extension analysis, polymerase chain reaction was used to amplify the fast and slow TnC mRNAs from cardiac and skeletal muscle. Analysis of the amplified products demonstrated the presence of significant amounts of slow TnC mRNA in the adult skeletal muscle. Key words: troponin C mRNA, skeletal muscle, cardiac muscle, polymerase chain reaction, developmental changes in muscle.
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BEREZOWSKY C., et BAG, J. 1992. Developmentally regulated troponin C mRNAs of chicken skeletal muscle. Biochem. Cell Biol. 70 : 156-165. La troponine C (TnC) rapide et la troponine C lente/cardiaque sont deux isoformes difftrentes. L'expression de ces isoformes dans les muscles squelettiques des verttbrts est soumise a une rkgulation dtveloppementale. Ainsi, nos etudes concernant leur expression ont porte sur les taux des d e w types de mRNA A I'ttat d'tquilibre. Nous avons Cgalement Cvalut I'efficacite de la traduction des mRNA des deux isoformes de la TnC au cours du dheloppement du muscle squelettique chez le poulet. Nous avons utilist diverses mtthodes afin de dtterminer les taux des mRNA a l'ttat d'tquilibre. Utilisant des fragments longs de 390 paires de bases (pb) et de 255 pb, obtenus respectivement de clones pleine longueur des cDNA de la forme rapide et de la forme lente de la TnC de poulet, nous avons d'abord dtveloppt des sondes sptcifiques des mRNA de la forme rapide et de la forme lente. La technique de buvardage du RNA a permis de montrer que le mRNA de la forme rapide de la TnC est l'isoforme prtdominante dans le muscle squelettique embryonnaire du poulet. Suite a l'tclosion, une quantitt importante du mRNA de la forme lente de la TnC s'accumule dans le muscle squelettique (pectoral). Quarante-trois semaines aprts llCclosion, le mRNA de la forme lente de la TnC est presqu'aussi abondant que celui de l'isoforme rapide. De plus, la majoritt des mRNA de la forme lente ainsi que de la forme rapide montrent une activite traductionnelle. Une seconde mtthode a permis une mesure plus fiable de la quantitt relative des mRNA de la forme lente et de la forme rapide de la TnC dans le muscle squelettique de poulet. Nous avons utilist une stquence commune, trts conservte, longue de 18 nucltotides et localiste vers la terminaison 5 ' de ces mRNA afin d'effectuer une analyse par extension d'amorce de ces d e w mRNA au cours d'une seule rtaction. Les rtsultats de ces analyses confirment la prtdominance du mRNA de la forme rapide de la TnC dans le muscle squelettique embryonnaire tandis qu'une accumulation importante du mRNA de la forme lente de la TnC est observte dans le muscle squelettique (pectoral) du poulet suite a I'klosion. En plus de l'analyse par extension d'amorce, l'amplification tlective (polymerase chain reaction) fut utilist pour amplifier les mRNA de la forme rapide et de la forme lente de la TnC du muscle squelettique ainsi que du muscle cardiaque. L'analyse des produits amplifits a dtmontrt la prtsence de quantitts importantes de mRNA de la forme lente de la TnC dans le muscle squelettique du poulet adulte. Mots elks : mRNA de la troponine C, muscle squelettique, muscle cardiaque, polymerase chain reaction, changements dCveloppementaux dans le muscle. [Traduit par la rtdaction]
Introduction The contractile proteins of skeletal and cardiac muscles exist in polymorphic forms, which are characteristic of the muscle fiber type. Gene expression of each member of an isoprotein family is regulated i n a tissue specific a n d developmental stage specific manner (Buckingham a n d Minty 1983). To understand how the switching o f synthesis f r o m one member of an isoprotein family t o the other occurs, we have chosen to examine this process during development o f chicken skeletal muscle using T n C as o u r model. T n C is one of the three troponin subunits that form a functional complex which regulates ABBREVIATIONS: TnC, TnT, and TnI, troponins C, T, and I; bp, base pair@);NP-40, Nonidet P-40; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; HBSS, Hanks' balanced salt solution; DMEM, Dulbeco's modified Eagle's medium; arcC; cytosine arabinoside; BSA, bovine serum albumin; BRL, Bethesda Research Labs; M-MLV, Moloney murine leukemia virus; PCR, polymerase chain reaction; Taq, Thermus aquaticus; a-Tm, a-tropomyosin. Printed in Canada / Imprime au Canada
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BEREZOWSKY AND BAG
the calcium-mediated contraction of striated muscle. While TnT and TnI have many known isoforms including fast, slow, and cardiac, TnC has only two known isoforms: fast and slow/cardiac (Buckingham and Minty 1983; Ebashi and Endo 1968; Gahlmann and Kedes 1990; Mannherz and Goody 1976). The slow TnC of embryonic skeletal muscle and the TnC of cardiac muscle have identical amino acid sequences and are considered to be the product of the same gene (Wilkinson 1980), whereas the fast TnC found only in adult skeletal muscle is the product of a different gene (Maisonpierre et al. 1987; Wilkinson 1980). In studies using monospecific antibodies to detect fast and slow TnC polypeptides, it was reported that during skeletal muscle development the slow or cardiac gene undergoes isogene switching and its expression is repressed in adult skeletal muscle (Toyota and Shimada 1981, 1983). In cardiac muscle, however, the cardiac TnC gene is expressed in embryonic as well as in adult heart (Toyota and Shimada 1981, 1983). The same gene (cardiac/slow) is thus regulated by different developmental programs in skeletal and cardiac muscle. The significance of developmental regulation of muscle proteins is not clear. It may be that the slow isoform of TnC is specific for sarcomere assembly during development of both skeletal and cardiac muscle in embryos and is replaced by the appropriate isoform in adult skeletal muscle. A single TnC isoform, however, appears to be sufficient during cardiac muscle development. It is probable that a large number of isoforms for contractile proteins may best fulfill the precise structural and kinetic requirements of each muscle fiber type at different stages of development. This implies that isogene switching may also be controlled by exercise and nerve influence (Swynghedauw 1986). To examine the changes in the expression of fast and slow TnC mRNAs during muscle development, the steady-state levels of these were measured. Furthermore, we have previously reported the presence of significant amounts of slow TnC mRNA in adult skeletal muscle (Berezowsky and Bag 1988) when previous studies using TnC antibody did not detect any slow TnC polypeptide in this muscle (Toyota and Shimada 1981, 1983). Therefore, have examined the subcellular distribution of TnC mRNA to determine if a significant amount of slow TnC mRNA is repressed from translation at any stage of muscle development in ovo or in cultured muscle cells. Materials and methods Subcellular fractionation and isolation of RNA The total cellular RNA from chicken skeletal and cardiac tissues was isolated using a modification of the guanidine - hot phenol procedure as previously described (Maniatis et al. 1982). To isolate polysomal and postpolysomal RNAs, the appropriate subcellular fractions were first isolated by a modification of the procedure as previously described by Bag and Sarkar (1975). Briefly, 1 g of the excised tissue was ground to a fine powder in liquid nitrogen and approximately 5 mL of lysis buffer (25 mM Tris-HC1 (pH 7.5), 250 mM NaCl, 5 mM MgCI,, 0.5% NP-40, 5 mM DTT, 50 pg cycloheximide/mL, 200 pg heparin/mL was added to the tissue. This mixture was homogenized in a dounce homogenizer with 20-30 strokes and examined under a microscope to ensure complete cell lysis. This mixture was then centrifuged at 8000 x g for 15 min at 1°C, and the postnuclear supernatant containing the polysomes was collected and adjusted to 500 pg heparin/mL. To pellet and isolate the polysomes, this postnuclear supernatant was centrifuged at 100 000 x g for 1 h at 1°C. The postpolysomal
157
supernatant was adjusted to 0.5% SDS and heated at 65°C for 5 min prior to RNA isolation. The polysomal pellet was dissolved in 10 mM Tris-HCI (pH 8.0) - 200 mM NaCl - 0.5% SDS. RNA was isolated from the polysomal and postpolysomal fractions by standard phenol-chloroform extractions as previously described (Maniatis et al. 1982). The final aqueous phase was adjusted to 0.2 M sodium acetate (pH 5.2) and the RNA was precipitated with 2.5 volumes of 100% ethanol at - 20°C. The RNA was pelleted at 8000 x g for 30 min, washed twice with 66% ethanol, dried briefly under vacuum, and dissolved in distilled H20. The concentrations of RNA in these samples were determined by measuring the absorbance at 260 nm and the integrity of the RNA in these preparations was determined by staining with ethidium bromide following agarose gel electrophoresis. The RNA samples were stored at - 80°C. Isolation of subcellular fractions from cells in culture Before harvesting, the cells were incubated with 20 pM emetine for 10 min at 37OC to prevent dissociation of polyribosomes during subcellular fractionation. The cells were washed four times (5 m ~ / 1 0 0 - c mplate) ~ with washing solution (130 mM NaCl, 5 mM KCI, 7.5 mM M MgCl,, 50 pg cyclohexamide/mL) and the cells were lysed on the plate by adding 4.0 mL of lysis solution (25 mM Tris-HC1 (pH 7.5), 250 mM NaCI, 5 mM MgCl,, 5 mM DTT, 0.5% NP-40, 50 pg cycIohexamide/mL, 200 pg heparin/mL. The cell lysate was prepared by homogenizing in a dounce homogenizer with 20-30 strokes. The cells were checked for complete lysis by examining the presence of nuclei free of cytoplasmic tags using a phase-contrast microscope. Cell culture Primary cultures of cardiac myocytes were prepared from cardiac tissue as previously described (Claycomb 1979; Malhotra and Bag 1987). Briefly, the cardiac tissue of 14-day-old embryonic chicken was treated with 0.1% collagenase and 0.1 Yo hyaluronidase for 15 min at 37OC. Multiple digestions were carried out. The cells obtained from the first two digestions were mostly fibroblasts and were discarded. Cells released by subsequent digestions (3-10) were pooled and the cardiac myocytes were separated from the fibroblasts by pelleting at 200 rpm (4 x g). Cardiac myocytes were plated at a desity of 2 x lo5 cells/cm2 and maintained in CMRL 1066 medium containing glutamine, 10% horse serum, 3% fetal bovine serum, and 40 pg insulin/mL (25 IU/mg, Sigma). The medium was supplementedwith 0.1 rnM 5 ' -bromo-2'-deoxyuridine during the first 24 h of incubation to select against dividing fibroblasts. Primary cultures of skeletal muscle cells were isolated from 12-day-old chicken embryos. In short, the skeletal muscle isolated from the breast and legs was minced into fine pieces with a scalpel. The minced tissue was placed in a sterile tube and washed twice with HBSS (Gibco) on ice. The tissue was then digested with 0.1% trypsin (Gibco) in HBSS at 37°C with agitation for 10-15 min. The digested tissue was filtered through a nylon membrane to remove any undigested material and this filtrate was then centrifuged at 1000 x g for 5 min at 4°C. The supernatant was removed and the pellet of skeletal muscle cells was suspended in DMEM containing 5% horse serum and 2% chick embryo extract (Gibco). The cells were plated on gelatin-coated plates at a cell density of 2 x 10' cells/cm2. The nutrient medium was supplemented with araC (Sigma) 24-48 h after plating to remove the dividing fibroblast cells. Gel electrophoresis of RNA and Northern blot analysis RNA was denatured in the presence of 50% formamide and 2.2 M formaldehyde before electrophoresis in 1% agarose gel containing 1.1 M formaldehyde and 10 mM sodium phosphate as previously described (Meinkoth and Wahl 1984). RNA from the agarose gel was transferred to a Zeta-probe blotting membrane (Bio-Rad) using 20 x SSC (1 x SSC: 0.15 M NaCl - 15 mM sodium citrate, pH 7.0). Following transfer, the membrane was baked at 80°C for 2 h and then washed in 0.1 x SSC - 0.5% SDS
BIOCHEM. CELL BIOL. VOL. 70.
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FAST TROPONIN C
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EcoRI
HinfI
HinfI
EcoRI
SLOW TROPONIN C
Hind111
Sau3AI
SalI
FIG. 1. Partial restriction map of slow and fast TnC cDNAs. Full-length fast and slow TnC cDNAs (Putkey et al. 1987; Reinach and Karlsson 1988) were subcloned into the multicloning site of vector pSPT18 (Pharmacia). Selected restriction sites are indicated by arrows. The 390-bp fast TnC HinfI and 255-bp slow TnC SadAI-SalI fragments are shown by hatched boxes. at 65°C for 30-60 min. Prehybridization was for 16-24 h at 65°C in 1% BSA (fraction 5) - 1 mM EDTA - 0.5 M NaH2P04 (pH 7.2) - 7% SDS. Hybridization was performed in the same buffer. A nick translation kit (IBI, Canada) was used to label probes with [ C Y - ~ ~ P ] ~ C (ICN; T P 3000 Ci/mM, 1 Ci = 37 GBq) and hybridization was performed for 48 h at 65°C. The membranes were washed two times for 30 min in 0.5% BSA (fraction 5) 1 mM EDTA - 40 mM NaH2P04 (pH 7.2) - 5% SDS at room temperature, followed by two washings at 65°C in 40 mM NaH2P04 (pH 7.2) - 1 mM EDTA - 1% SDS for 30 min. The washed filters were exposed to Kodak X-AR film using Cronex intensifying screens for 48-72 h at - 80°C.
200 U M-MLV reverse transcriptase (BRL) at 42°C for 2 h, with addition of another 200 U M-MLV reverse transcriptase after 1 h of incubation. The reaction mixture was adjusted to 0.2 M sodium acetate (pH 5.2) and nucleic acids were precipitated with 2.5 volumes of 95% ethanol using 5 pg tRNA (wheat germ tRNA, Sigma) as the carrier. The primer extension reaction products were then analyzed by electrophoresison a 10% polyacrylamide sequencing gel containing 7.7 M urea (Maxam and Gilbert 1980). The gel was fixed in 10% acetic acid (v/v) - 10% methanol (v/v) for 15 rnin and rinsed with distilled H 2 0 before drying. The dried gels were exposed to Kodak X-AR film using Cronex intensifying screens for 24-48 h at - 80°C.
Slot blotting of DNA and primer extension analysis of RNA Slot blotting of DNA was performed on a Minifold I1 slot-blot apparatus (Schleicher and Schuell) according to the manufacturers directions. Briefly, the DNA sample was suspended in 400 pL of 10 mM Tris-HC1 (pH 7.0) - 1 mM EDTA. The sample was adjusted to 0.3 N NaOH and incubated at 65°C for 30 min before neutralizing with an equal volume of 2 M ammonium acetate (pH 7.0). The sample was immobilized under vacuum onto a Zetaprobe membrane (Bio-Rad) prewetted in 1 M ammonium acetate (pH 7.0). The membrane was processed for prehybridization and hybridization as described for Northern blotting of RNA. Primer extension analysis of RNA samples was performed by a modification of the method previously described (Maisonpierre et al. 1987). The primer employed was an 18-base oligomer (5'CCCAGCATCCTCATCACC3'),which is antisense to a sequence common to both fast and slow TnC mRNAs (Putkey et al. 1987; Reinach and Karlsson 1988). This primer would hybridize towards the 5 '-end of both messages (see Fig. 6). The 18 oligomer was 5'-end-labelled using [Y-~~PIATP (ICN; 7000 Ci/mM) and polynucleotide kinase (BRL) (Maniatis et al. 1982). The labelled oligomer was purified from unincorporated radioisotope by gel filtration using a Bio-Gel P-4 column (Bio-Rad). The purified oligomer (50 ng) was added to the RNA sample (5 pg) in buffer containing 5 mM NaH2P04 (pH 7.2) and 5 mM EDTA, in a volume of 10 pL. The sample was denatured at 85°C for 6 min and annealed on ice for 10 min. The nucleic acid from the reaction mixture was precipitated and brought to a final volume of 20 pL in a buffer containing 50 mM Tris-HC1 (pH 8.3 at 42"C), 8 mM MgCI,, 30 mM KCl, 1 mM DTT, and 2 mM each of four dNTPs. Extension of the radiolabelled primer was carried out using
Synthesis of cDNA and its amplifcation by PCR Reverse transcription of both fast and slow TnC mRNAs was performed with the common antisense oligomer (Fig. 6) as the primer. One microgram of total cellular RNA was mixed with 100 ng of primer and heated for 6 min at 85°C. The reaction mixture was then cooled to 50°C and annealed for 10 min. Reverse transcription was performed as previously described (Sambrook et al. 1989) with 200 U MMLV reverse transcriptase (BRL) at 50°C for 30 min. Synthesis of double-stranded cDNA and subsequent amplification of the product was done with 100 ng of an oligomer primer specific for fast or slow TnC mRNA (Fig. 9) and 1 U Taq DNA polymerase (BRL) as previously described (Sambrook et al. 1989). Amplification was performed for 25-30 cycles in a Perkin Elmer Cetus thermocycler (1 cycle: 70 s at 94"C, 120 s at 5S°C, and 180 s at 72°C). The PCR amplification product was analyzed by electrophoresis in nondenaturing 8% acrylamide gel. The expected size DNA was electroeluted from the gel and further characterized by digestion with appropriate restriction enzymes. The electroeluted DNA was cloned into pBluescript I1 KS + / (Stratagene) after treatment with the Klenow fragment of DNA polymerase I and polynucleotide kinase (BRL). The DNA insert of the clone was sequenced using the dideoxychain termination method of Sanger (Sanger et al. 1977) employing a Sequenase V2.0 kit (United States Biochemical Corporation).
Results Subcellular distribution of TnC and tropomyosin mRNAs Results of a number of earlier studies (Mikawa et al. 1981; Toyota and Shimada 1981) have shown that the embryonic
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BEREZOWSKY AND BAG
FIG. 2. Analysis of the specificity of TnC cDNA probes. Purified full-length cDNA inserts of both fast and slow TnC cDNA clones were blotted onto Zeta-probe (Bio-Rad) nylon membrane as described in Materials and methods. Fast and slow specific probes (see Fig. 1) were nick-translated and hybridized as described under Materials and methods. (i) Probed with the 390-bp HinfI fragment of fast TnC cDNA; (ii) probed with the 255-bp Sau3AISan fragment of slow TnC cDNA. Full-length fast (Sk) and slow (Cd) TnC cDNA inserts were immobilized for hybridization: (a) 2.5 ng; (b) 5.0 ng DNA. The arrows indicate the positions of 2.5 ng of pBR322 DNA. skeletal muscle of chicken consists of both fast and the slow isoforms of the TnC polypeptide, whereas the adult chicken skeletal muscle is composed entirely of fast TnC. Previous studies from our laboratory, however, have shown that a significant amount of slow TnC mRNA is present in the adult chicken skeletal muscle (Berezowsky and Bag 1988). It is possible that the discrepancy between these results could be due to failure of translation of slow TnC mRNA in adult skeletal muscle. We have, therefore, first examined if the TnC mRNAs are efficiently translated at different stages of development of skeletal muscle. This was analyzed by examining the distribution of TnC mRNAs between the translationally active polysomal and inactive postpolysomal fractions of skeletal breast (pectoralis) muscles. The nonhomologous quail slow TnC cDNA clone used in our previous studies (Berezowsky and Bag 1988), however, hybridized to both fast and slow TnC mRNAs (results not shown). Therefore an attempt was made to obtain probes specific for fast and slow TnC mRNAs. The full-length chicken slow and fast TnC cDNAs (Putkey et al. 1987; Reinach and Karlsson 1988) were also found unsuitable for this purpose. Particularly the full-length slow TnC cDNA showed some cross-hybridization to the fast TnC mRNA (results not shown). Analysis of the homology of nucleotide sequences between different regions of the fast and slow TnC mRNAs using the DNAsis sequence comparison program (Hitachi Biologicals) suggested that the 390-bp-long HinfIdigested restriction fragment of the 5'-end of fast TnC cDNA clone and the 255-bp-long Sau3AI- and San-digested restriction fragment of the 3 '-end of slow TnC cDNA clone (Fig. 1) could be used as fast and slow specific probes, respectively. The fast probe exhibited a maximum of 38% homology within some region of the slow TnC mRNA. On the other hand, the slow specific fragment showed a maximum of 50% homology within some region of the fast TnC mRNA. T o determine if hybridization under stringent conditions would allow detection of a specific TnC mRNA using these fragments as probes, hybridization of nick-translated probes with full-length inserts of both fast and slow TnC cDNAs
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FIG. 3. Northern blot analysis of fast and slow TnC mRNAs of skeletal and cardiac muscles. Poly(A)-rich RNA from skeletal and cardiac muscles were isolated from the total cellular RNA samples using oligo(dT)-cellulose chromatography as previously described (Maniatis et al. 1982). One microgram of each poly(A)rich RNA was electrophoresed in an agarose gel and used for RNA blotting. Skeletal (Sk) and cardiac (Cd) muscle poly(A)-rich RNAs were probed with either the 390-bp fast TnC fragment (Sk' and Cd') or with the 255-bp slow TnC fragment (Sk and Cd). was examined by slot-blot analysis. The result presented in Fig. 2 shows that no detectable cross-hybridization of these probes was observed. Furthermore, Northern blot analyses of skeletal and cardiac muscle mRNAs (Fig. 3) show that the fast TnC cDNA fragment did not hybridize to the TnC mRNA of cardiac muscle. In contrast, the slow TnC cDNA fragment produced a strong signal when RNA from either skeletal or cardiac muscle of a 40-week-old bird was used (Fig. 3). Since there was no cross-hybridization between the slow 255-bp fragment and the full-length insert of fast TnC cDNA in the slot-blot analysis, it was concluded that the hybridization of the slow TnC cDNA probe with the skeletal muscle RNA was due to the presence of significant level of slow TnC mRNA in skeletal muscle. These studies show that the 390- and 255-bp restriction fragments of fast and slow TnC cDNA clones can be used to determined the steadystate levels of fast and slow TnC mRNAs, respectively. Therefore, we have used these fragments to measure the levels of fast and slow TnC mRNAs in the polysomal and postpolysomal fractions of skeletal muscle at various stages of development. The result of Northern blot analysis of skeletal RNA samples probed with the fast and slow TnC mRNA specific probes is shown in Fig. 4. Nearly all of the fast TnC mRNA of 14-day-old embryonic skeletal muscle was in the polysome fraction (Fig. 4, panel i). However, in the 21- and 43-weekold birds, a large proportion (21-26%) of the fast TnC mRNA was found in the postpolysomal fraction. When the same RNA samples were probed with the slow TnC cDNA fragment (Fig. 4, panel ii), no detectable level of the slow TnC mRNA was found in the breast muscle of 14-day embryo. At 21 weeks posthatching, detectable levels of the slow TnC mRNA were found in both polysomal and post-
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34
I4
A
a
B
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FIG. 4. Subcellular distribution of mRNAs in chicken skeletal muscle. Northern blot analysis of chicken skeletal muscle RNA was performed as described in Materials and methods. Fifteen micrograms of the polysomal RNA (upper case letters) and an equivalent amount of the postpolysomal (lower case letters) RNA were used for analysis. (i) RNA samples were probed with the 390-bp fast TnC cDNA fragment; (ii) RNA samples were probed with the slow 255-bp TnC cDNA fragment. (A, a) Fourteen day embryo; (B, b) hatchling; (C, c) 2 weeks posthatching; (D, d ) 21 weeks posthatching; (E, e) 43 weeks adult. (iii) RNA samples were probed with chicken a-Tm cDNA. (A, a) Fourteen day embryo; (B, b) hatching; (C, c) 2 weeks posthatching; (D, d ) 43 weeks posthatching. The autoradiograms were scanned and the peak area was measured. The numbers above each lane represent the area corresponding to the signal in an arbitrary unit.
polysomal fractions (Fig. 4, panel i, lanes D and d). In 21and 43-week-old birds, 22-35% of slow TnC mRNA was also found in the translationally repressed postpolysomal fraction. Since there was no detectable hybridization of the slow TnC probe to the RNA samples from the breast muscle of 14-day embryo and hatchling, it is unlikely that the signals detected in the breast muscle of older birds was due to crosshybridization of the probe with the fast TnC mRNA. These results (Fig. 4 and Table 1) therefore show that detectable levels of slow TnC mRNA is present in the adult skeletal muscle. Furthermore, no significant difference in the distribution of fast and slow TnC mRNAs between the translationally active polysomal and repressed postpolysomal fraction was observed. The major portion (64-78s) of both TnC mRNAs was found in the polysomal population.
1992
Therefore, preferential repression of slow TnC mRNA translation was not detected. Our results presented here show that in the adult chicken breast muscle approximately 40% of total TnC mRNA consists of the slow isoform. In contrast, the fast TnC mRNA was the only TnC isoform detectable in the embryonic skeletal muscle. Accumulation of the slow TnC mRNA began following hatching. Our results also suggest that in contrast to the embryonic skeletal muscle where TnC mRNA was not detectable in the translationally repressed postpolysomal fraction, 22-36% of cytoplasmic TnC mRNAs of adult skeletal muscle were found in this fraction. To examine if this was specific for TnC mRNAs, we analyzed the subcellular distribution of mRNA for another contractile protein of the thin filament. The result of these analyses using an a-Tm specific probe (MacLeod 1981) is shown in panel iii of Fig. 4. The a-Tm mRNA was found to be translated efficiently in the skeletal muscle of up to the 2-week-old chicken. In adult birds, however, approximately 36% of a-Tm mRNA was also found in the translationally repressed postpolysomal fraction (Fig. 4 and Table 1). This pattern of subcellular distribution of TnC mRNAs and a-Tm mRNA was reproducible in several experiments using different RNA samples. Approximately 10% difference in the subcellular distribution profile was noticed between experiments. In adult birds, the presence of a substantial portion of the mRNAs in the repressed fraction may be due to their general inability to translate mRNAs efficiently. Also, the possibility of dissociation of polyribosomes during the isolation procedures cannot be completely ruled out. All the procedures, however, for subcellular fractionation included cycloheximide to prevent dissociation of polyribosomes. Developmental changes in the distribution of TnC mRNAs between polysomal and postpolysomal fractions in whole tissues prompted us to study whether similar changes occurred in cardiac and skeletal muscle cells in culture. In these analyses we used the quail cDNA probe because it hybridizes efficiently to both fast and slow TnC mRNAs. The result of the steady-state levels of polysomal and postpolysomal cytoplasmic TnC mRNA(s) of skeletal and cardiac muscle cells at various times after plating is shown in Fig. 5. The results show that in cardiac muscle cells, there was no significant change in the steady-state level of TnC mRNA between 4 and 14 days after plating of cardiac myocytes. Furthermore, TnC mRNA was not detected in the postpolysomal fraction (Fig. 5, panel i). In skeletal myotubes (Fig. 5, panel ii) the level of TnC mRNA was maximum at 14 days after plating, and like the cardiac myocytes, no TnC mRNA was detectable in the repressed fraction. It appears that TnC mRNA was translated efficiently in both cardiac and skeletal muscle cells in culture during the period of sampling in our studies. Therefore, translation of TnC mRNA in these cells in culture resembled embryonic and early neonatal tissues in chicken. In examining the relative steady-state levels of fast and slow TnC mRNAs during development of skeletal muscle using Northern blot analysis, one problem is variation in the specific activity between probes. Since both fast and slow messages migrate in the same region of 1-1.5% agarose gels and thus are not resolveable as two distinct bands, separate Northern blots need to be processed to determine the levels of fast and slow TnC mRNA for each sample. In addition
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TABLE1 . Subcellular distribution of mRNAs at various stages of skeletal muscle development Distribution of mRNA (%)
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/13/14 For personal use only.
Slow TnC
Fast TnC
Develo~mentstage
Polysomal
Free'
Polysomal
14-day embryo Hatching 2-week posthatching 21-week posthatching 43-week posthatching
-
-
100 100 100 78 74
100 64 78
-
36 22
Freea
Polysomal
-
100 100 90 ND 64
NOTE:These values were obtained by scanning the RNA blot shown in Fig. 4. ND, experiments not performed. 'Free refers to the postpolysomal fraction.
the possibility of hybridization of slow TnC probe with the fast TnC mRNA and vice versa cannot be completely ruled out in RNA-blot analysis. To circumvent these problems we used primer extension analysis using a common primer to determine the steady-state levels of both fast and slow TnC mRNAs in a single reaction. The nucleotide sequences of fast and slow TnC mRNAs were compared to find a common oligonucleotide sequence that could be used for primer extension of both mRNAs. A highly conserved 18-nucleotide-long sequence close to the 5 '-end and common to both fast and slow TnC mRNAs of chicken, mouse, and human has been detected (Parmacek and Leiden 1989; Putkey et al. 1987; Reinach and Karlsson 1988). This nucleotide sequence is shown in Fig. 6. These conserved 18 nucleotides are present between nucleotide 148 and 165 of slow TnC mRNA and between nucleotide 177 and 194 of fast TnC mRNA. Therefore, an antisense oligonucleotide to this conserved region would hybridize to both fast and slow TnC mRNAs and could produce primer extended products of 194 and 165 nucleotides in length, respectively. This possibly would allow us to determine the relative levels of both mRNAs using a single reaction. If a 5 '-end-labelled oligomer is used as a primer, the extended products could be quantified by using autoradiography. We initially examined if conditions employed for primer extension was within the linear range of response to the concentration of mRNAs. The result of these analyses is shown in Fig. 7. Increasing concentrations of poly(A)+ mRNAs from breast and cardiac muscle of 14-day-old chick embryos were used for primer extension. The results show that breast muscle mRNAs produced a 194 nucleotide long major primer extended product (lanes A'-C'). On the other hand, cardiac mRNA produced one major band (lanes A-C) and a distinct minor band. As judged from the molecular size markers (not shown), this major band appeared to be larger than 165 nucleotides in length. To determine the correct primer extended products of both fast and slow TnC mRNAs, the two bands shown by arrows (Fig. 7) were sequenced using the method described by Maxam and Gilbert (1980). Analysis of the nucleotide sequence confirmed that the 194-nucleotide-long primer extended product was derived from the fast TnC mRNA, and the band with an apparent molecular size of 185 nucleotides produced by primer extension of cardiac mRNA was in fact the correct product derived from the cardiac/slow TnC mRNA. Furthermore, the lower
aTIt,
-
22 26
Freea
10 ND 36
-, no detectable signal was found;
FIG. 5. Subcellular distribution of TnC mRNA in cardiac (i) and skeletal (ii) muscle cells in tissue culture. Northern blot analysis of 15 pg polysomal (upper case letters) and an equivalent amount of postpolysomal (lower case letters) RNA from cardiac myocytes (A-C, a-c) and skeletal myotubes (Af-C', a'-c'). RNA samples shown in panels i and ii were probed with quail TnC cDNA. (A, a, A ' , a') Four days; (B, b, B ' , b') 7 days; (C, c, C ' , c') 14 days after plating. Each of the lanes was scanned and the area under each peak was determined. The value corresponding to the signal in each lane is presented above the lane using an arbitrary unit.
molecular weight band corresponding to the cardiac TnC mRNA, found in the primer extension product of breast muscle mRNA, was also sequenced and found to be derived from the cardiac/slow TnC mRNA. It was concluded, therefore, that the slower migration of the cardiac/slow TnC mRNA derived primer extension product relative to the size
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BIOCHEM. CELL BIOL. VOL. 70, 1992
SLOW TROPONIN C 20 148 165 655 . . . ATG .../ / ...ggggaaGGTGATGAGGATGCTGGGgcagaa .A [ 1651
