0022-1514/92/$3.30 The Journal of Histochemistry and Cytochemisny Copyright 0 1992 by The Histochemical Society, Inc.

Vol. 40, No. 10, pp. 1547-1517, 1992 Printed in UXA.

Original Article

I Fast Myosin Light Chain Expression in Chicken Muscles Studied by In Situ Hybridization’ RUDOLF BILLE’IER, 2y3 MARIUS MESSERLI, EVA WEY, ADRIAN PUNTSCHART,3 KRISTIN JOSTARNM: HANS M. EPPENBERGER, and JEAN-CLAUDE PERRIARD Institute of Cell Biology, Swiss Federal Institute of Technology, Honggerberg, CH-8093 Ziiricb, Switzerland Received for publication February 6, 1992 and in revised form April 6, 1992; accepted May 11, 1992 (2A2183).

We have studied the fiber type-specificexpression of the fast myosin light chain isoforms LC If, LC 2f, and LC 3f in adult chicken muscles using in situ hybridization and twodimensional gel electrophoresis. Type I1 (fast) fibers contain all threefast myosin light chain “As; T p I and III (slow) fibers lack them. The myosin light chain patterns of twodimensional gels from microdissectedsingle fibersmatch their mRNA signals in the in situ hybridizations. The results confirm and extend previous studies on the fiber type-specific distribution of myosin light chains in chicken muscles which used specific antibodies. The quantitative ratios between protein and mRNA content were not the same for all three fast myosin light chains, however. In bulk muscle samples, as well as in single fibers, there was proportionally less LC 3f accumulated for a given mRNA concentration than LC If or LC 2f. Moreover, the ratio between LC 3f mRNA and pro-

Introduction In situ hybridization of cellular mRNA is becoming an increasingly popular method to study differential expression of closely related protein isoforms, mainly because of the relative ease with which specific probes can be generated once the nucleic acid sequences coding for an isoprotein family are known. In vertebrates, the approach works particularly well with embryos (e.g., Izpisiia-Belmonte et al., 1991) or brain sections (‘Maishi et al., 1991; Brandt et al., 1987). In the various fiber types of skeletal muscles, isoforms of myofibrillar proteins are the basis for many of their different physiological properties. The “classic” fiber type concept is based on differences in the susceptibility of the myofibrillar ATPase to acid or alkali (Brooke and Kaiser, 1970; Guth and Samaha, 1969). The ATPase stain reflects the myosin heavy chain isoform composition of a given

Supported by the Swiss National Science Foundation (grant 3.497-0.86). Correspondence to: R. Billeter, Inst. of Anatomy, Univ. of Berne, Biihlstr. 26, CH-3000 Berne 9, Switzerland. Present address: Institute of Anatomy, University of Berne, CH-3000 Berne 9, Switzerland.

*

tein was different in samples from muscles, indicating that

LC 3f is regulated somewhat differently than LC If and LC 2f. In contrast to other in situ hybridization studies on the fiber type-specific localization of muscle protein “As, which reported the RNAs to be located preferentially at the periphery of the fibers, we found all three fast myosin light chain mRNAs quite evenly distributed within the fiber’s cross-sections, and also in the few rare fibers which showed hybridizationsignals several-fold higher than their surrounding counterparts. This could indicate principal differences in the intracellular localization among the mRNAs coding for various myofibrillar protein families. ( J Histochem Cytochem 40:1547-1557, 1992) In situ hybridizations;Chicken skeletal muscle; Muscle fiber types; Myosin light chains; Two-dimensional gel electrophoresis; Single fiber analysis; Gene expression.

KEY WORDS:

fiber (Staron, 1991; Billeter et al., 1981; Gauthier and Lowey, 1977, 1979). The isoforms of all the myofibrillar proteins investigated thus far differ in their mRNA sequences. Many of these isoforms are encoded on different genes, others are generated by differential splicing (Breitbart et al., 1987). In situ hybridizations with isoform specific probes for slow and fast myosin heavy chain mRNA (Hesketh et al., 1991; Brand et al., 1990; Aigner and Pette, 1990; Diu and Eisenberg, l988,1990,1991a,b,c),as well as troponin I mRNA (Koppe et al., 1989), have shown that these mRNAs are located in their respective fiber types and absent in the others. Our study extends these observations to the fast myosin light chain isoforms of chicken skeletal muscles. In vertebrate adult muscle, the fast myosin light chains LC If, LC 2f, and LC 3f have primarily been located in Type I1 (fast twitch) fibers (Crow et al., 1983; Mikawa et al., 1981; Gauthier and Lowey, 1979; Pette and Schnez, 1977). Human m. vastus lateralis is one of an increasing number of exceptions;in this muscle, LC If occurs also in Type I (slow twitch) fibers in coexistence with the slow isoforms LC 1s and LC 2s (Billeter et al., 1981). Most light chain isoforms are encoded on separate genes (reviewed in Barton and Buckingham, 1985), except for the fast alkali light chains LC If and LC 3f, which are transcribed from the 1547

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on June 15, 2015

1548

BILLETER, MESSERJJ, WEY, PUNTSCHART, J O S ” D T ,

same gene. These two proteins differ from each other only at the NH2 terminus, where, in the chicken, LC 3f has eight specific amino acids and LC If 49. They both have identical COOH termini of 144 amino acids. The mRNAs of LC If and LC 3f are generated from two different promotors via a series of specific, constitutive splicing steps (Gallego and Nadal-Ginard, 1990; Billeter et al., 1988; Nabeshima et al., 1984; Periasami et al., 1984; Robert et al., 1984). Our in situ hybridizationsshow the “As coding for the three fast myosin light chains to be located exclusively in the fast (Type 11) fibers of chicken muscles. This is in agreement with the protein work done utilizing isoform specific myosin light chain antibodies (Crow et al., 1983,1986;Gauthiet et al., 1984; Gauthier and Lowey, 1979). A semiquantitative comparison of RNA and protein concentrations of bulk muscle samples indicated that LC 3f is regulated in a slightly different way from LC If and LC 2f. When we compared our results with the in situ hybridization studies that localized other mRNAs in adult skeletal muscle, we found the myosin light chain ”As distributed evenly in the muscle fiber crosssections, not preferentially at their periphery. Unexpectedly, we found a small number of Type I1 fibers which had several-foldhigher levels of all three fast myosin light chain mRNAs than their surrounding counterparts.

Materials and Methods Mzlrcles Seven-weekold male broiler chickens (Vedette, a Dwdw commercialbroiler strain, originating from a cross of Plymouth Rock x Cornish, bred by ISA, Lyon, France) were obtained from Kneuss (Magenwil,Switzerland),where they were fed a hormone-free broiler diet (13.4 MJlkg; 20.0% crude protein) while being raised in loose-floorhousing according to the guidelines provided by the supplier. They were sacrificed by decapitation, the desired muscles [m. anterior latissimus dorsi (ALD). m. satorius (Sart), m. adductor medialis (MA), m. gastrocnemiusmedialis, m. pectoralis maior, m. semitendinosus, m. semimembranosus] were immediately excised, selected regions cut into strips about 3 x 10 mm, and frozen in freon cooled by liquid nitrogen. For in situ hybridizations and ATPase staining, 12-pm cryostat sections were cut, thawed onto microscope slides, that were treated with aminoakylsilane(Rentrop et al., 1986), and stored at -20’C for up to 2 weeks.

Isolation of Total RNA Total RNA was isolated according to Reisine et al., (1985) from 10-100 mg of frozen muscle tissue of areas adjacent to the samples used for cryostat sectioning.

Nortbern Blots Northern blots were carried out according to Sambrook et al., (1989), using RNA from m. anterior latissimus dorsi, m. gastrocnemius medialis, and m. pectoralis maior. Twelve pg total RNA per lane were applied onto 1.5% formaldehyde gels. The gels were blotted onto nitrocellulose filters by the capillary elution method. These filters were hybridized with 32P-labeled LC lf. 2f. or 3f anti-sense probes (see below), using the same conditions as for in situ hybridization, except that dextran sulfate was reduced to 10 ng per ml to avoid background problems (Anderson and Young, 1985). Exposure time was 2-3 days.

EPPENBERGER, PERRIARD

Probes We used synthetic RNA probes in our experiments. They were transcribed from constructs with pSP6S or 64 plasmids (Promega; Madison, WI). Depending on the orientation of the insert, either RNA with a sequence complementary to the mRNAs is synthesized with SP6 polymerase (anti-sense RNA probe, used for specific hybridization),or RNA with a sequence identical to the mRNA (sense probe, used as negative control). On Northern blots with adult clcken muscle RNA, the probes bound specifcallyto bands of the expected lengths (Figure 2).

LC lf/3f Probes. The probes for LC If and LC 3f were derived from full-length cDNA clones (Billeter et al., 1988)(Figure 1). Hae 111fragments from specific regions of the cDNAs were inserted in both orientations into the Sma I site of the plasmid pSP65. The inserts were 198 BP in the case of the LC If probe and 74 BP in the case of LC 3f. Their G/C content was 5 5 and 53%, respectively. LC 2f Probe. The probe specific for chicken LC 2f mRNA originated from a double-stranded synthetic oligodeoxynucleotide which contained nucleotides 48-106 of the sequence published by Reinach and Fischman (1985) (see Figure 1). linked to the 17 essential nucleotides of the bacteriophage T7 RNA polymerase promotor (Milligan et al., 1987) by two dC residues on the template strand. Its borders consisted of the sticky ends of an Eco R I site at the 5’ and Bam HI at the 3’ end, which were used for subcloning into pSP64. From the resulting construct, the LC 2f-specific anti-sense probe could be transcribed with T7 polymerase and the sense (control) probe with SP6 polymerase. The G/C content of the LC 2f specific insert was 59%. In vitro RNA synthesis of the probes used for in situ hybridizations was performed according to Melton et al., (1984) using [35S]-UTP(1380 CilmM) (NewEngland Nuclear; Boston, MA) as radioactive label. This resulted in average specific activities of 2 x 10’ cpmlE. Probe synthesis and all subsequent steps were performed in solutions to which fresh DTT had been added to 20 mM to minimize nonspecific 31S binding. In situ hybridization experimentswith an LC If-specificanti-senseprobe transcribed from the single-strandedtemplate (Milligan et al., 1987)failed. These transcriptions yielded transcripts shorter than full length, ending primarily at positions of the radioactive nucleotide, which was present at concentrationsbelow K, in ow reactions (data not shown). T7 polymerasedirected transcription of the anti-sense probe from the double-stranded oligodeoxynucleotide as well as from the plasmid subclone, on the other hand, resulted in about equal amounts of full-length transcripts and small oligonucleotides(smaller than 18 residues), which could not be completely removed by columns of Sephadex with pore sizes between G 25 and G 200. In situ hybridization experiments with our LC 2f anti-sense probe therefore gave distinctly higher background than experimentswith LC If or LC 3f anti-sense probes which were transcribed by Sp6 polymerase (Figures, 3, 4, and 5 ) . 32P-labeledprobes were generated in the same way using [ 32P]-GTP (800 Ci/mM) (Amersham; Poole, UK), except for the LC 2f probe used in the Northern blot experiment presented in Figure 2, where [32P]-UTP was used. T7 transcription of the LC 2f probe with [32P]-GTPresulted in extremely varying yields, presumably because the transcript starts with a G j stretch, which leads to polymerase detachment when GTP is the limiting nucleotide.

In Situ Hybridizations In situ hybridizations were carried out essentially according to Chesselet et al. (1987). Cryostat sections (10-12 pm) were fixed for 15 min in 4% paraformaldehydein PBS followed by incubation steps in 0.25% acetic anhydride in 0.1 M triethanolamine. in 0.1 M Trislglycine, pH 7, and subsequent dehydration. Hybridization was carried out for 3-4 hr in 20 p1 hybridization solution at 45’C under parafilm in a sealed chamber moistened

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on June 15, 2015

1549

MYOSIN LIGHT CHAIN EXPRESSION IN CHICKEN MUSCLES

Figure 1. Sequences used for the generation of specific chicken fast myosin light chain RNAprobes. ThecDNAsof thechicken fast myosinlight chains are schematically depicted with the positions of restrictionsites for Hae 111 and Fnu4H1 indicated. The specific as well as the common regions of LC 11and LC 3f mRNA are labeled differently. Bars above the maps mark the fragments used as probes. In the case of LC I f and LC 31 mRNA, they were Hae Ill fragments; in the case of LC 2f,an oligodeoxynucleotide was synthesizedwhich included nt 48 to 106 of the published sequence. The numbers above the bars show the cutting sites of the 5' and 3' end of the oligodeoxynucleotide, respectively. These fragmentswere inserted into the plasmids pSP65 or 64 (see text), from which the synthetic RNA probes used in these experiments were transcribed.

102 -301

r--"' '

H

H I Hoe Ill F = FnuLHl

@

1

@ 2

3

&A-

HF

U L C l f speciltc 0LC3f specific t3 LC2f specific c7 LClf/3f common reglon Probes

F

1

101

F

H

LClf (1041- 1172b)

F

LC3f

(856 - 983 b)

4220 \\

-

FHF

F FH

H

F HF

LC2f

(689 b)

RNAse concentration, were empirically determined in hybridization experiments similar to those of Lawrence and Singer (1985). using [''PIprobes on slides containing sections of m.pectoralis maior (consisting of Type I1 fibers with fast myosin light chains only) and the posterior part of m. adductor medialis (consisting of Type 111 slow tonic fibers only with no fast myosin light chains) (Zhang et al., 1985).

Autoradiograp by The sections were dehydrated in a series of ethanol steps with 300 mM ammonium acetate instead of water. They were then dipped in NTB 2 Emulsion (Kodak; Rochester. NY) and exposed at 5'C for 1-4 weeks. The autoradiographs were developed in Kodak D 19 developer, followed by a short wash in 1% acetic acid and fiition in Hypam (Ilford; Mobberley, England) diluted 1:4. Incubation in water instead of 1% acetic acid often caused the emulsion to detach from the aminoalkylsilane-treatedslides. The slides were air-dried and mounted in Kaiser's gelatin (Merck; Darmstadt. Germany).

0

1 2 3

RNAse Mapping

.28 S

( .

F

3620 1 , 1

with 4 x SSC. The hybridization solution consisted of hybridization buffer (4 x SSC, 40% formamide, 10% dextran sulfate, 1 x Denhardt's solution, 1 pglpl E. coli tRNA, 1 pglpl calf thymus DNA. and 20 mM DTT) with the specific RNA probes in concentrations of 2-5 ngl20 pl (cquivalent to 200.000-500.000 cpmlpl). Specificanti-senseRNA probes(see above) were used to detect the "As. Controls included hybridizationswith sense probes, with hybridization buffer alone, or sectionspre-treated with RNAse before hybridization. Washeswere done in 1 x SSC. 50% formamide (LC 3f and LC 2f probes) or in 2 x SSC, 50% formamide (LC If probes) for 20 min at 60°C. followed by digestion with RNAse A (Boehringer; Mannheim. Germany) at 0.5 pglml for 1 hr at 37'C. After another specific wash at 60'C for 20 min. the sections could be left overnight in 2 x SSC, 0.05% Triton X-100 at room temperature. if necessary. All wash solutions contained fresh 0.1 M DTT. The optimal temperatures for hybridization and washes, as well as the

1 2 3

I

1

FH

F

RNAse mapping was carried out according to Melton et al. (1984). Total RNA (4 pg) prepared from muscle pieces adjacent to the blocks used for in situ hybridization were co-precipitated with 100,000 cpm of the 32Plabeled anti-sense probes specific for LC If. LC 2f, and LC 3f. Hybridization was carried out at 52'C overnight in 80% formamide, 40 mM PIPES, 400 mM NaC1, 1 mM EDTA. The samples were subsequently digested with RNAse A (33 pglml) and T1 (2 pglml) and the products separated on a 10% polyacrylamide sequencing gel. The resulting bands were scanned two dimensionally with an optical scanner and the peaks integrated. The ratios presented in lible 1 were calculated after comction for the differentamounts of 32P-labeledG residues in the probes.

oo-'8s

Figure 2. Northern blots of RNA from three different chicken muscles hybridized with the fast myosin light chain RNA probes, illustratingprobe specificity. (a) Hybridization with anti-sense probe specific for LC I f mRNA. (b) Hybridization with anti-sense probe specific for LC 2f mRNA. (c) Hybridization with antisense probe specific for LC 31 mRNA. Lanes 1 represent total RNA from m. anterior latissimus dorsi. Lanes 2 represent, total RNA from m. gastrocnemius medialis. Lanes 3 represent total RNA from m. pectoralis major. The much weaker signals for fast myosin light chain mRNAs in ALD reflect the small proportion of Type IIfibers in this muscle. Markers indicate the positionof the gel slot, 28 S. and 18 S RNA in the different gel runs.

Isolation and Epigenetic Labeling of Fragments from Single Muscle Fibers Fragmentsfrom selected fibers (Figure 6) were isolated according to Staron and Pette (1986) from a 100-pm cryostat section of MA sequential to the ones used for in situ hybridization or histochemical staining. The 100-pm sectionwas lyophilizedat -35'C and the desired fibersweremicrodissected at x 50 magnification. The proteins of these single fiber fragments were ''C-labeled by reductive methylation (Billeter et al., 1981; Jentoft and Dearborn, 1979). The reaction was terminated by the addition of 11 p1

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on June 15, 2015

BILLETER, MESSERLI, WEY, PUNTSCHART, JOSTARNM: EPPENBERGER, PERRIARD

1550

.

Figure 3. In situ hybridization of chicken m. anterior latissimus dorsi (pars anterior) (ALD) with probes specific for the fast myosin light chain mRNAs. In the autoradiographs, the fiber circumferences are indicated in black ink, redrawn from phasecontrast photographs obtained from the same sections. Fiber types are indicated as derived from the myofibrillar ATPase stain. (a) Hybridization with anti-sense probe specific for LC 11mRNA. (b) Hybridization with anti-sense probe specific for LC 2f mRNA. (c) Hybridization with anti-sense probe specific for LC 31mRNA. (d) Hybridization with sense probe identical to LC 3f mRNA serving as a negative control. (e) Myofibrillar ATPase staining after pre-incubation at pH 4.45. (f) NADH-tetrazolium reductase stain. The last myosin light chain mRNAs are located in Type II(fast twitch) fibers. The "slow tonic" libers in ALD differ from the "slow twitch" fibers in, e.g., m. sartorius, by their myosin type. They are called Type 111 (Barnard et al.. 1982). I, slow twitch; II, fast twitch; 111, slow tonic. Bar = 100 um.

,.

.

H20, 4 p1 b-mercaptoethanol, and 20 pI 10% Nonidet P 40 during an incubation at 37°C for 10 min. Five pI undiluted ampholytes. pH 4-6.5 (Pharmacia; Piscataway, NJ) were then added and 57 mg urea dissolved in the sample before loading it onto the first-dimension isoelectric focusing gel.

Two-dimensional Gel Electrophoresis Two-dimensional gel electrophoresis was performed essentially according to OFarrell et al. (1975). Isolated fragments from single fibers were processed as described above. For a semiquantitative estimate of the myosin light chain content in whole muscle blocks, one to four 40-pm cryostat sections serial to the ones used for in situ hybridization were dissolved by addition of 15 p1 hot SDS sample buffer (Laemmli. 1970) and immediate incubation in boiling water for 3 min. After removal of non-solubilized material by ccntrifugation. 16 pI H20. 4 1 1 b-mercaptoethanol, and 20 pl 10% Nonidet P 40 were added. the sample cooled to room temperature and 57 mg urea

and 5 pI ampholytes, pH 4-6.5. dissolved in it before loading onto the first-dimension gel. Isoelectric focusing was carried out using ampholytes in the p H 4-6.5 range with 0.01 M histidine as cathode buffer and 0.01 M glutamic acid as anode buffer. The second dimension was a 15% polyacrylamide gel. After separation on two-dimensional gels, the proteins were transferred onto nitrocellulose membranes by semi-dry blotting. The ''C-methylated proteins from the single fiber fragments were visualized by autoradiography on pre-flashed Fuji RX film. Exposure time was 4-6 weeks. The proteins from.the whole cross-sections were stained with amido black and the spots scanned on an optical scanner.

Histochemistry Myofibrillar ATPase was carried out as described for chicken muscle by Butler and Cosmos (1981) and Phillips and Bcnnct (1984). Acid pre-incubation was at pH 4.3 and 4.45. Alkali pre-incubation was at pH 10.3 and 10.4. The fibers were typed according to Barnard e t al. (1982).

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on June 15, 2015

MYOSIN LIGHT CHAIN EXPRESSION IN CHICKEN MUSCLES

1551

Figure 4. In situ hybridization of chicken m. adductor medialis (pars anterior) (MA) with probes specific for the fast myosin light chain mRNAs. (a) Hybridization with antisense probe specific for LC I f mRNA. (b) Hybridization with anti-sense probe specific for LC 21 mRNA. (c) Hybridization with antisense probe specific for LC 31 mRNA. (d) Hybridization with sense probe identical to LC 31 mRNA serving as a negative control. (e) Myofibrillar ATPase staining after preincubation at pH 4.45. (1) NADH-tetrazolium reductase stain. Fiber types are indicated based on acid and alkali (not shown) stability of their ATPase. The fast myosin light chain mRNAs are located in type II (fast twitch) fibers. The fiber marked by the arrowhead is one of the rare Type II fibers which yielded much stronger in situ hybridization signals than its surrounding counterparts. Abbreviations as in Figure 3. Bar = 100 pm.

Results Probe Specz3city

Correlation Between Histochemical Fiber Types ana' Fast Myosin Light Chain mRNAs

The synthetic RNA probes used in the experiments presented here were all directed against the 5' portions of the chicken fast myosin light chain mRNAs (Figure 1). Their specificity is shown with Northern blots (Figure 2), which show that the anti-sense probes specifically bound to RNAs in the expected size ranges (about 1100 to 1200 B for Lc lf, 700 B for L c 2f and 900-1000 B for Lc 3f mRNA). Strong signals were produced by the RNAs from the two muscles containing a significant amount of fast (Type 11) fibers (m. pectoralis, m. gastrocnemius medialis) and weak ones by the RNAs from ALD, which contains only few Type I1 fibers. In the case of chicken LC If and LC 3f mRNAs, unusually broad bands are sometimes encountered on Northern blots, stemming from doublets that originate from the use of two different poly-A addition sites spaced 112 B apart. These sites are used in about equal proportions (Billeter et al., 1988). Additional evidence for the specificity of our probes comes from the fact that they yielded fragments of the expected sizes in the RNAse mapping experiments (not shown).

To compare the fast myosin light chain expression pattern with the histochemical fiber types of adult chicken muscles, serial sections were either stained for myofibrillar ATPase or subjected to in situ hybridizations with the myosin light chain anti-sense RNA probes. Examples of such series are presented in Figures 3-6. In these experiments, the presence of the mRNAs coding for fast myosin light chains (LC If, LC 2f, and LC 3f) could be demonstrated in Type I1 (fast twitch) fibers only. Our anti-sense probes bound strongly to all Type I1 fibers in the anterior portions of m. anterior latissimus, dorsi (ALD) and the anterior most portion of m. adductor medialis (MA) (Figures 3a-3c and 4a-4c), for example. Binding was reproducibly moderate to weak, but above background, in the Type I1 fibers of the middle and white portion of m. sartorius (Sart, white portion; Figure 5 ) , whose sections were processed on the same microscope slide. In the slow fibers (Type I er Type 111) of either muscle, however, the signals could not be distinguished from the experimental background, which is repre-

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on June 15, 2015

BILLETER, MESSERLI, WEY, PUNTSCHART, JOSTARNDT, EPPENBERGER, PERRIARD

1552

Figure 5. In situ hybridization of a representative section from the deep (white) part of chicken m. sartorius(Sart) with probes spa cific for the fast myosin light chain mRNAs. (a) Hybridization with anti-sense probe specific for LC If mRNA. (b)Hybridizationwith anti-sense probe specific for LC 2f mRNA. (c)Hybridization with anti-sense probe specific for LC 31 mRNA. (d) Hybridizationwith sense probe identicalto LC 31mRNA sewing as a negative control. (e) MyofibrillarATPase staining after pre-incubation at pH 4.45. (1) NADH-tetrazolium reductase stain. Fiber types are indicated.The fiber marked by the arrowhead is one of the rare fibers that gave several-fold stronger signals than its surrounding counterparts. Bar = 100 wm.

sented by the binding of the LC 3f sense probe in this series (Figures 3d, 4d, and 5d). Similar results were obtained with sections from m. semitendinosus, m. gastrocnemius medialis, and the red (superficial) porTable 1. Ratios of myosin light chain mRNAs andproteim in bulk muscle .ramblesa Muscle

m . sartorius, whole m . sartorius. red (superficial) part m . sartorius, white (deep) part m. semitendinosus m. adductor medialis

&A ratio IC1 f:LC2f:LC3f

Protein ratio LC 1 f:LCZf:LC3f

0.55:1:0.7

1: 1:0.2

0.55:1:0.65

1:1:0.15

0.65:l:1.05 0.60:1:0.75 0.65:1:0.60

1:1:0.3 1:1:0.2 1: 1:0.03

a The numbers represent relative ratios of the fast myosin light chain proteins and their mRNAs from bulk smples taken from areas adjacent to the sections used for in situ hybridization. There is considerably more LC 3f mRNA per LC 3f protein compared with LC If. The ratio of LC 3f protein to LC 3f mRNA in MA is lower than in the other muscles.

tion of m. sartorius, using different sense strand probes which produced similar background levels (data not shown).

A Few Rare Fibers Yield Exceedingly Strong In Situ Hybridization Reactions with Probes for AZZ Fast Myosin Light Chain mRNAs A small number of fibers (less than 1%)produced signals that were several-fold higher than the reactions of the surrounding Type I1 fibers or Type I1 fibers in adjacent cross-sections processed on the same slide. Examples of such fibers are shown in Figures 4-6. The fiber shown in the white part of Sart (arrowhead in Figure 5 ) is particularly impressive because it contrasts with the somewhat lower signals of normal Type I1 fibers in Sart compared to Type I1 fibers of MA or ALD. The frequency of such fibers varied among different muscles and chickens. We found a small number of them in most crosssections from the anterior portion of MA in six different chickens. In cross-sectionsof one whole m. sartorius, 53 such fiben were found in a total of 25,000 fibers, of which 79% were Type 11; in the cross-

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on June 15, 2015

MYOSIN LIGHT CHAIN EXPRESSION IN CHICKEN MUSCLES

1553

Figure 6. Comparison of in situ hybridization reactions with the myosin light chain protein patterns of selected fibers from the anterior most portion of MA. (1-6) Myosin light chain regions of two-dimensional gels obtained from 14C-labeled fragments of fibers which were dissected from a 100-wm cryostat section adjacent to the ones presented in the figure. They correspond to the fibers numbered 1-6 on sections a to d. The myosin light chains are labeled as identified by their position on the 2D gels. (a) Hybridization with anti-senseprobe specific for LC I f mRNA. (b) Hybridization with anti-sense probe specific for LC 31 mRNA. (c) Hybridiza tion with sense probe identical to LC 31 mRNA serving as a negative control. (d) Myofibrillar ATPase staining after pre-incubation at pH 10.3. Fiber types are indicated. Note the higher content of LC 3f mRNA and protein in fiber No. 3. Bar = 100 pm.

2

1

3

LClS

LClf

t

LClf

*

28

21

2f

b

31

0

31 1

6

5

4

LCIS

LClf 0

8

21

0

sections of a sartorius from another chicken, we found none. In ALDs from three different chickens, only one such fiber was found. All the fibers yielding very strong signals with the three fast light chain RNA probes were histochemically Type 11. Most of them had diameters and cross-sectionalshapes like their neighboring “normal” fibers (Figures 4-6); a minority of them were very small and rounded (not shown).

Comparison of In Situ Hybridization Reactions with the Myosin Light Chain Patterns of Individual Fibers in M. Adductor Medialis In an attempt to demonstrate experimentally that the same fibers that have fast myosin light chain mRNAs also contain the corresponding proteins, we isolated fragments of selected fibers out of freeze-dried 100-pm sections serial to the 12-pm sections used for

31

LClS 11

-

28

-

4

.

28 2f

- 4

3f

in situ hybridization, such as the fibers from the MA presented in Figure 6. The proteins of these fragments were epigenetically labeled with [ l4C]-forma1dehyde and subsequently separated on two-dimensional gels. The 2D gel patterns, indicative of the myofibrillar isoform profile of the fiber, could then be compared to the in situ hybridization signals of the same fiber in the sequential cross-sections. Fiber Numbers 2 and 5 are Type I fibers, as deduced from their weak ATPase reaction after alkali pre-incubation (Figure 6d). As judged by the position of the myosin light chain spots on the gel, they both contain the slow isoforms LC Is and LC 2s but lack fast light chains. This is in agreement with the hybridization signals obtained with the LC If and LC 3f anti-sense RNA probes, which were indistinguishable from background. Type I1 fibers (Numbers 1, 3, 4 and 6), as expected, contained all three fast myosin light chain proteins. They also all yielded distinct signals for LC If and 3f mRNA in the hybridization experi-

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on June 15, 2015

1554

BILLETER, MESSERLI, WEY, PUNTSCHART, JOSTARNDT, EPPENBERGER, PERRIARD

ment (Figures 6a and 6b). Fiber Number 6 also contained a small amount of the slow myosin light chains. Fiber number 3 is another fiber whose in situ hybridization signal with LC If and LC 3f is stronger than the signals of the surrounding Type I1 fibers. The difference between the LC 3f mRNA signal of this particular fiber and the signal of its surroundingcounterparts is distinctly larger than the LC If signal differences, however. Correspondingly, the LC 3f protein content is higher than the LC 3f content of fiber Number 1, for example. This strongly staining fiber also displayed a slightly stronger alkali-stable ATPase (Figure 6d), suggesting it to be of Type IIB.

Fast Myosin Light Chain mRNA and Protein Ratios in Tissue Samples from Adult Chicken Muscles To obtain more quantitativeinformation on the correlationbetween the fast myosin light chain proteins and their mRNA concentrations, we isolated total RNA from bulk samples adjacent to the sections used for in situ hybridization. The RNA was subjected to RNAse mapping with the anti-sense probes for all three fast myosin light chain “As in the same reaction, after which the resulting gel bands were scanned, which enabled us to calculate the proportions of the fast myosin light chain mRNAs. The myosin light chain protein content of these muscle regions was estimated from scans of two-dimensional gel electropherograms from extracts of adjacent 40-pm cross-sections. As can be seen in Table 1, the different “mixed” leg muscles examined have similar ratios of the fast myosin light chain mRNAs and proteins. If protein and “A ratios are compared, however, it is obvious that there is several-fold more LC If protein per mRNA accumulated than LC 3f. When compared with LC If and 3f, the mRNA-to-protein ratios for LC 2f lie between the values for LC If and LC 3f. In our experiments, the ratio between LC 3f protein and mRNA was not constant among different muscles. MA had distinctly less LC 3f protein accumulated than the other muscles analyzed, despite similar relative mRNA levels.

Discussion In this study, we attempted to localize the mRNAs coding for the fast myosin light chains in the fiber types of adult chicken skeletal muscles. As expected, these ”As were found exclusively in Type I1 fibers, thus confirming and extending a number of studies on the fiber type-specific distribution of the myosin light chain proteins. In addition, we were able to demonstrate that the same fibers that contain fast myosin light chain mRNAs also contain the proteins. Our observation is compatible with the hypothesis that the fiber type-specific expression of myofibrillar proteins is primarily achieved by transcriptional control although it offers no proof for this hypothesis. A more quantitative analysis of bulk RNA and proteins from adjacent muscle samples indeed revealed discrepancies in the ratios of mRNA:protein for LC 3f among different muscles, indicating that other mechanisms are involved as well. In contrast to the findings of other authors, who described the RNAs coding for different proteins to be preferentially located in

the peripheral areas of the muscle fibers, we found all three fast myosin light chain RNAs evenly distributed within the fiber crosssections, and also in the few rare fibers that generated hybridization signals several-foldhigher than their surroundingcounterparts. This could indicate principal differences in the intracellular localization among the mRNAs coding for various myofibrillar protein families.

Fiber Tjpe-speczj5cLocalization of Fast Myosin Light Chain mRNAs and their proteins Most of the studies presented thus far on the fiber type-specific localization of the different myosin light chain proteins in chicken skeletal muscles offer data on the same muscles used in this study.

MA (m. adductor medialis, also named m. adductor profundus). In the anterior region of this principally “slow tonic” muscle (Barnard et al., 1982), a mixture of fast and slow fibers has been found (Zhang et al. 1985; Crow et al., 1983). In contrast to Zhang et al. (1985), we found a majority of the slow fibers in the muscle’s most anterior part to be of Type I instead of Type 111, based on the nomenclature of Barnard et al. (1982), because after acid preincubation their myofibrillar ATPase reaction was stronger than that of Type 111fibers (Figure 4e) and after alkali pre-treatment it was extinguished (not shown). Type I1 fibers in MA were of IIA subtype, based on their strong NADH tetrazolium reductase stain (Barnard et al., 1982) (Figure 4f). The in situ hybridizationspresented in Figures 4a-4d match the results obtained by Crow et al. (1983), who used an antibody that was directed to the LC lf/3f common region and could therefore not distinguish between LC If and LC 3f in the cross-sections. In MA, this antibody labeled fast (Type 11) fibers but not slow (Type 111 or I) fibers. The experiment presented in Figure 6 goes a step further, since we could show that the same fibers reacting positively for fast myosin light chain mRNA also contain fast myosin light chain proteins, whereas non-reactive fibers do not. Fiber Number 6 in Figure 6 has small amounts of slow myosin light chains in addition to the fast isoforms. This is also in agreement with these authors, who described some MA fibers to react with an antibody to the slow myosin LC Is as well as with an antibody to fast myosin LC lf/3f. ALD (m. anterior latissimus dorsi). This muscle consists mainly of Type 111fibers with a few small Type IIA fibers located in the most anterior part of the muscle, which is represented in Figure 3. These IIA fibers contain fast myosin light chain mRNA, as shown in Figures 3a-3d. In contrast to Gauthier et al. (1984) and Crow et al. (1983),we could not detect any in situ hybridization signals that would correspond to the LC If or LC 3f antibody reactions in some slow fibers (mainly IIIB) (Gauthier et al., 1984). Using two-dimensional gel electrophoresisto separate the proteins of single (presumablyType 111) fibers from ALD, Mikawa et al. (1981) could not detect fast myosin light chains either. This discrepancy could be due to age or strains of the chickens. Alternatively, one might consider that techniques using antibodies are more sensitive than gel electrophoresis or techniques demonstrating mRNA signals, such as our in situ hybridizations.

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on June 15, 2015

1555

MYOSIN LIGHT CHAIN EXPRESSION IN CHICKEN MUSCLES

Sart (m. sartorius). The overall pattem of in situ hybridizations with probes specific for the fast myosin light chain mRNAs (Figure 5) corresponds to the myosin light chain pattem presented in Crow and Stockdale(1986). 2d gel analysis of the proteins from untyped, single sartorius fibers show either the slow (LC 1s and LC 2s) or all three fast light chains occurring in a given fiber but no other combination (Mikawa et al., 1981; and our unpublished results). The different NADH tetrazolium reductase staining intensities of the Type I1 fibers in Sart, as presented in Figure 5 , indicate the presence of Type IIA and IIB fibers. In this muscle as well as in m. semitendinosus, which also has large proportions of Type IIA and IIB fibers (not shown), we did not see any differences in signal intensity between Type I1 fiber subtypes, such as for LC 3f in fiber Number 3 of Figure 6. The weaker fast myosin light chain mRNA signals of Type I1 fibers in the middle and deep (white) portion (Figure 5 ) of m. sartorius as well as m. semitendinosus (not shown) when compared with the signals in MA and ALD (Figures 3 and 4) could reflect lower turnover rates, as have been measured in muscles with high fast fiber proportions (Gregory et al., 1990; Garlick et al., 1989). In situ hybridization experimentsperformed with other isoformspecific probes have demonstrated fiber type specific localization for the mRNAs coding for slow (p) myosin heavy chain (Dix and Eisenberg, 1988,1990,1991a; Aigner and Pette, 1990; Brand et al., 1990), a fast type myosin heavy chain (Diu and Eisenberg, 1991b), and the mRNAs for troponin I isoforms (Koppe et al., 1989). These data as well as ours are compatible with the hypothesis that the expression of myofibrillar protein isoforms is primarily controlled at the level of transcription; they provide no proof for this, however. Transcriptional control has been shown to play a role in the coordinated expression of myofibrillar proteins during myogenesis (Cox et al., 1991), but this study and others (e.g., Gregory et al., 1990)also offer evidence for regulation by additional mechanisms. In none of our experiments with adult chicken muscles did we find fibers that reacted with only two of our probes but not with the third, containing, e.g., exclusively LC If and LC Zf or LC 3f and LC 2f mRNA, nor did we find such patterns in the protein gels of single fibers. LC If and LC 3f are therefore co-expressed in adult chicken muscle fibers. The resolution of our method would not have been sufficient to detect selective synthesis of LC If or LC 3f mRNA in neighboring nuclei of a muscle fiber, however.

Intracellular Localization of Fast Myosin Light Chain mRNAs In the majority of our experiments (illustrated in Figures 3,4, and 6), the autoradiographygrains were evenly distributed over the fibers reacting positively with the fast myosin light chain RNA probes. This indicates-within the resolution limits ofthe method-an even distribution of the myosin LC mRNAs within the fibers. Only occasionally did we detect stronger labeling at the periphery of positive fibers, for example, as shown in Figure 5a for the Type I1 fibers in the middle part of Sart. Our observations contrast with the results obtained by Dix and Eisenberg (l988,1991a,c), Aigner and Pette (1990), Brand et al. (1990), and Hesketh et al. (1991). all ofwhom used anti-senseprobes specific for slow (p) myosin heavy chain mRNA in rat and rabbit

muscles. These authors found the myosin heavy chain mRNAs located preferentially in the periphery of the fibers. Similar results were obtained by Dix and Eisenberg (1991b), who used a fast myosin heavy chain mRNA probe in rabbit muscles, Kelly et al. (1991), who localized carbonic anhydrase I11 mRNA in rat muscles, and Koppe et al. (1989), who utilized probes specific for the different troponin I isoform mRNAs on rat muscles. We have employed the same hybridization methods as the authors of the above studies, the major differences being the type of mRNA studied (myosin light chain vs myosin heavy chain, troponin I and carbonic anhydrase 111) and the animals (chicken vs rodents). The structure of chicken muscle is very similar to that of rodent muscle, however; we know of no feature that could account for the preferential peripheral localization of RNA in rodent but not in chicken muscle fibers. It is more likely that the myosin light chain mRNAs are principally more evenly distributed in muscle codingfor, e.g., myosin heavy chain or tropofibers than the “As nin. Further experiments involving the localization of RNAs of different protein families in muscle fibers of the same species are necessary to clarlfy this point.

Semiquantitative Correlation Between Fast Myosin Light Chain Proteins ana’ Their mRNAs in BUM Muscle Samples The bulk analysis of samples from muscles used also for in situ hybridization (Table 1) indicate that LC 3f is regulated somewhat differentlyfrom LC If and LC 2f. We found constant but different ratios between accumulated mRNAs and proteins for LC If and LC 2f, but not for LC 3f, where in MA the amount of LC 3f protein was much lower compared with the other muscles examined, despite similar mRNA content. These results corroborate the studies on “fast to slow” transformation of rabbit and rat muscles by electrical stimulation, in which it was shown that the synthesisrate of the myosin light chains correlates with the levels of their mRNAs (Bar et al., 1989, Kirschbaum et al., 1989),except for LC 3f. LC 3f (and possibly the other myosin light chains as well) is therefore also regulated posttranscriptionally. Additional evidence for posttranscriptional regulation of LC 3f comes from Roy and Sarkar (1982), who investigated LC 3f expression during the development of chicken pectoral muscles. In particular, there is always more LC If protein per mRNA accumulated compared with LC 3f, as has been shown by Pluskal and Sreter (1983) as well as Heilig and Pette (1983) for rabbit, Biir et al. (1989) for rat, and Roy and Sarkar (1982) for chicken muscles. Two mechanisms for posttranscriptional control of LC 3f have been proposed. Kirschbaum et al. (1989)provided evidence for regulation of LC 3f levels by changes in its protein degradation rate during electrostimulation of adult rabbit muscles. Roy and Sarkar (1982) suggested that LC 3f expression is regulated by some form of translational control during chicken muscle development.

A Few Rare Fibers Show Very Stmng Reactions with All Three Myosin Light Chain RNA Pr0be.r An unexpected finding in our study was the occurrence of fibers that showed very strong hybridization signals (Figures 4, 5 , and

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on June 15, 2015

1556

BILLETER, MESSERLI, WEY, PUNTSCHART, J O S ” D T ,

6). They were rare, however, totalling less than 1% of the fibers in any of the muscles sampled, being absent in some. Almost all of these particular fibers yielded very strong signals with all three of the fast myosin light chain RNA probes. In addition, no preferentially peripheral location of the hybridization signal could be detected in these fibers. Examination of serial cross-sections showed that they displayed strong reactivity over distances of at least several hundred p m . This indicates that these high myosin light chain mRNA concentrations are probably the product of many nuclei. O u r observations with adult chicken muscles provided no indications o n the cause of this phenomenon. Fibers with high levels of B-myosin heavy chain m R N A at the myotendinous junction as well as the mid-belly region have been f o u n d in rabbit muscles immobilized in a stretched position (Dix and Eisenberg 1990, 1991a,c). T h e muscles of the chickens we obtained were not subjected to experimental treatment, however, nor do we have indications that their diet (hormone-free broiler diet) or farming circumstances (loose-floor housing) could give rise to increased degenerationhegeneration or repair of muscle fibers.

Acknowledgments We are indebted to Dr M. B Chesseletf o r a thorough introduction to the in situ hybridization technique, to Dr D. Sassoon for additional suggestions on the in situ hybridizations, to P m f M . Lezzi f o r instrumental help in setting up the autoradiography, andto Drs H.U. Affolterand D. Schumperli f o r logistic as wellas technical help concerning the generation of specipc probes.

Literature Cited Aigner S, Pette D (1990): In situ hybridization of slow myosin heavy chain mRNA in normal and transforming rabbit muscles with the use of a nonradioactively labeled cRNA. Histochemistry 95:11 Anderson MLM, Young BD (1983): Quantitative filter hybridization. In: Hames BD, Higgins SJ, eds. Nucleic acid hybridization. A practical approach. Oxford, IRL Press, 75 B’dr A, SimoneauJ-A, Pette D (1989): Altered expression of myosin lightchain isoforms in chronically stimulated fast-twitch muscle of the rat. Eur J Biochem 178:591 Barnard EA, LylesJM, PizzeyJA (1982): Fibre types in chicken skeletal muscles and their changes in muscular dystrophy. J Physiol 331:333 Barton PJ, Buckingham M (1985): The myosin alkali light chains and their genes. Biochem J 231:249 Billeter R, Heizmann CW, Howald H, Jenny E (1981): Analysis of myosin light and heavy chain types in single human skeletal muscle fibers. Eur J Biochem 116:389 Billeter R, Quitschke W, Paterson BM (1988): Approximately 1 kilobase of sequence 5’ to the two myosin light-chain lf13f gene cap sites is sufficient for differentiation-dependentexpression. Mol Cell Biol 8:1361 Brand T, Schaper J, Munkel B, Sharma HS, Bleese N, Schaper W (1990): In situ hybridization study on myosin heavy chain expression in human heart failure. In: Marechal G, Carraro U, eds. Muscle and motility. Proc. XMth conference. Hanover, Intercept, 175 Brandt SJ, Niedel JE, Bell RM, Young WS (1987): Distinct patterns of expression of different protein kinase C mRNAs in rat tissues. Cell 49:57 Brooke MH, Kaiser KK (1970): Muscle fiber types: how many and what kind? Arch Neurol 23:369

EPPENBERGER, PERRIARD

Breitbart RE, Andreadis A, Nadal-Ginard B (1987): Alternative splicing: a ubiquitous mechanism for the generation of multiple protein isoforms from single genes. Annu Rev Biochem 36:467 ButkrJ, Cosmos E (1981): Dfirenuauon ofthe avian latissimus dorsi primordim:analysis of fibre tvpe expression using the myosin ATPasc histochemical reaction. J Exp Zoo1 218:219 Chesselet MF. Weiss L, Wiinschell C, Tobin AJ, Affolter HU (1987): Comparative distribution of “A’s for glutamic acid decarboxylase, tyrosine hydroxylase and tachikinins in the basal ganglia. An in situ hybridization study in the rodent brain. J Comp Neurol 242:125 Crow MT, Olson PS, Stockdale FE (1983): Myosin light chain expression during avian muscle development. J Cell Biol 69:736 Crow MT, Stockdale FE (1986): Myosin expression and specializationamong the earliest muscle fibers of the developing avian limb. Dev Biol 113:238 Cox RD, Weydert A, Barlow D, Buckingham ME (1991): Three linked myosin heavy chain genes clustered within 370 kb of each other show independent transcriptional and post-transcriptional regulation during differentiation of a mouse muscle cell line. Dev Biol 143:36 Dix DJ, Eisenberg BR (1991a): Distribution of myosin mRNA during development and regeneration of skeletal muscle fibers. Dev Biol 143:422 Diu DJ, Eisenberg BR (199lb): Expression ofa fast myosin heavy chain mRNA in individual rabbit skeletal muscle fibers with intermediate oxidative capacity. Anat Rec 230:52 Diu DJ, Eisenberg BR (1991~):Redistribution of myosin heavy chain mRNA in the midregion of stretched muscle fibers. Cell Tissue Res 263:61 Dix DJ. Eisenberg BR (1990): Myosin mRNA accumulation and myofibrillogenesis at the myotendinous junction of stretched muscle fibers. J Cell Biol 111:1885 Dix DJ, Eisenberg BR (1988): In situ hybridization and immunocytochemistry in serial sections of rabbit skeletal muscle to detect myosin expression. J Histochem Cytochem 36:1519 Gallego ME, Nadal-Ginard B (1990): Myosin light-chain 113 gene alternative splicing: cis regulation is based upon a hierarchical compatibility between splice sites. Mol Cell Biol 10:2133 Garlick PJ, Maltin CA, Baillie AGS, Delday MI, Grubb DA (1989): Fiber type composition of nine rat muscles 11. Relationship to protein turnover. Am J Physiol 237:E828 Gauthier GF, Lowey S (1979): Distribution of myosin isoenzymes among skeletal muscle fiber types. J Cell Biol 81:lO Gauthier GF, h y S (1977): Polymorphism of myosin among skeletal muscle fiber types. J Cell Biol 74:760 Gauthier GF, Ono RD, Hobbs AW (1984): Curare-induced transformation of myosin pattern in developing skeletal muscle fibers. Dev Biol 105:144 Gregory P, Gagnon J, Essig DA, Reid SK, Prior G, Zak R (1990): Differential regulation of actin and myosin isoenzyme synthesis in functionallyoverloaded skeletal muscle. Biochem J 265:525 Guth L, Samaha FJ (1969): Qualitative differences between actomyosin ATPase of slow and fast mammalian muscle. Exp Neurol 25:138 Heilig A, Pette D (1983): Changes in transcriptional activity of chronically stimulated fast twitch muscle. FEBS Lett 151:211 Hesketh J, Campbell B, Loveridge N (1991): Myosin heavy-chain mRNA is present in both myofibrillar and subsarcolemmal regions of muscle fibers. Biochem J 279309 Izpisua-Belmonte JC, Tickel C. Doll6 P,Wolpert L, Buboule D (1991): Expression of the homeobox Hox-4 genes and the specification of position in chick wing development. Nature 350:583 Jentoft N, Dearborn DG (1979): Labelling of proteins by reductive methylation using sodium cyanoborohydride. J Biol Chem 294:4359 Kelly CD. Carter ND, De Boer P, Jeffery S, Moorman AFM, Smith A (1991):

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on June 15, 2015

1557

MYOSIN LIGHT CHAIN EXPRESSION IN CHICKEN MUSCLES

Detection of CAI11mRNA in skeletal muscle and liver by in situ hybridization. J Histochem Cytochem 39:1243 Kirschbaum BJ, SimoneauJA, Bar A, Barton P, Buckingham M, Pette D (1989): Chronic stimulation-induced changes of myosin light chains at the mRNA and protein levels in rat fast-twitch muscle. Eur J Biochem 179:23 Koppe RI, Hallauer PL, Karpati G, Hastings KEM (1989): cDNA clone and expression analysis of rodent fast and slow skeletal muscle troponin I “As. J Biol Chem 26414327 Laemmli UK (1970): Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680 LawrenceJB, Singer RH (1985): Quantitative analysis of in situ hybriditation methods for the detection of actin gene expression. Nucleic Acids Res 13:1777 Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR (1984): Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 12:7035 Mikawa T, Takeda S, Shimizu T, Kitaura T (1981): Gene expression of myofibrillar proteins in single muscle fibers of adult chicken: micro twodimensional gel electrophoretic analysis. J Biochem 89:1951 MilliganJF, Groebe DR, Witheress GW, Uhlenbeck OC (1987): Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res 1523783 Nabeshima Y, Fujii-Kuriyama Y,Muramatsu M, Ogata K (1984): Alternative transcription and two modes of splicing result in two myosin light chains from one gene. Nature 308:333 O’Farrell PH (1975): High resolution two-dimensional electrophoresis of proteins. J Biol Chem 2504007 Periasami M, Strehler EE, Garfinkel LE, Gubits RM, Ruiz-Opazo N, Nadal-Ginard B (1984): Fast skeletal muscle myosin light chains 1 and 3 are produced from a single gene by a combined process of differential RNA transcription and splicing. J Biol Chem 259:13595 Pette D, Schnez U (1977): Myosin light chain patterns of individual fast and slow-twitch fibres of rabbit muscles. Histochemistry 54:97 Phillips W, Bennett MR (1984):Differentiation of fiber types in wing muscles during embryonic development: effect of neural tube. Dev Biol 106:457

Pluskal MG, Sreter FA (1983): Correlation between protein phenotype and gene expression in adult rabbit fast twitch muscles undergoing a fast to slow fiber transformation in response CO electrical stimulation in vivo. Biochem Biophys Res Commun 113:325 Reinach FC, Fischman DA (1985): Recombinant DNA approach for defining the primary structure of monoclonal antibody epitopes. The analysis of a conformation-specific antibody to myosin light chain 2. J Mol Biol 181:411 Reisine T, Rougon G, Barbet J, Affolter H-U (1983): Corticotropin-releasing factor-induced adrenocorticotropinhormone reIeax and synthesis is blocked by incorporation of the inhibitor of cyclic AMP-dependent protein kinase into anterior pituitary tumor cells by liposomes. Proc Nad Acad Sci USA 82:8261 Rentrop M, Knapp B, Winter H, Schweizer J (1986): Aminoalkylsilanetreated glass slides as support for in situ hybridization of keratin cDNAs to frozen tissue sections under varying fixation and pretreatment conditions. Histochem J 18:271 Robert B, Daubas P, Akimenko MA, Cohen A, Gardner I, Guenet IL, Buckingham M (1984): A single locus in the mouse encodes both myosin light chains 1 and 3, a second locus corresponds to a related pseudogene. Cell 39:129 Roy RK, Sarkar S (1982): Correlation between the protein and mRNA levels for myosin light chains and tropomyosin subunits during chick fast muscle development in vivo. FEBS Lett 149:22 Sambrook J, Fritsch EF, Maniatis T (1989): Molecular Cloning. A laboratory manual. Cold Spring Harbor, NY,Cold Spring Harbor Laboratory Press Staron RS (1991): Correlation between myofibrillar ATPase activity and myosin heavy chain composition in single human musde fibers. Histochemistry 9621 Staron RS, Pette D (1986): Correlation between myofibrillar ATPase activity and myosin heavy chain composition in rabbit muscle fibers. Histochemistry 8639 likaishi T, Saito N, Kuno T, ”aka C (1991): Differential distribution of the mRNA encoding two isoforms of the catalytic subunit of calcineurin in the rat brain. Biochem Biophys Res Commun 174:393 B a n g Y, RushbrookJI, Shafiq SA (1985): Avian adductor profundus muscle: characterization of a pure slow tonic region by histochemical, monoclonal antibody and peptide mapping studies.J Muscle Res Cell Mod1 6:333

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on June 15, 2015