283.

TECHNIQUES FOR STUDY OF SYNTHE'SIS F. C. PARRISH, JR. IOWA STATE UNIVERSITY ......

A study of skeletal muscle protein biosynthesis requires a number of both simple and sophisticated techniques because it involves a study of cellu- lar subunits and molecules and their reactions. Many of these techniques h?-ve already been successfully used to acquire a considerable body of knowledge about muscle and ultrastructure. Consequently, adaptation of these techniques provides a strong potential for obtaining some very inter- esting and illuminating information about muscle protein biosynthesis and development. Other aids in the study of muscle protein biosynthesis hwe been the knowledge supplied by the molecular biologist on the biosynthetic mecha- nism of cells and on the behavior of actin and myosin in solution. Recent research, utilizing many of the same techniques as those used for the study of protein chemistry and structure, has provided us with some very profound information about myof ibrillogenesis of mammalian skeletal muscle. Naturally, the subject of myofibrillogenesis is of much interest and concern to the muscle biologist and meat scientist because the of the myofibril are the ones that most directly affect muscle growth and development, contraction, rigor mortis, meat tenderness, water binding, emulsification and human nutri- tion. If we are able to make substantial improvement in the quantitative, qualitative, and nutritive characteristics of meat we must turn to the use of techniques that will yield information on how muscle proteins are synthesized and formed into meat at the cellular and molecular level. With knowledge gained at these levels we can then begin to regulate those mechanisms and compounds affecting muscle growth and composition.

The purpose of this paper is to present information about certain preparative and analytical techniques used in the study of myofibrillar protein biosynthesis. The kind of information obtained, rather than the mechanics of the technique, will be emphasized. Also, the techniques listed are not intended to be dl inclusive, usually only one investigator will be cited, although these same or similar techniques have been used by other researchers, and in some instances some of these techniques could be classified as both preparative and analytical. Furthermore, many specific techniques are embodied within a general technique. First of all, I would like to describe very briefly several general techniques. For a detailed description, one should refer to the materials and methods section of the original paper. Then I would like to specifically refer to the morphological techniques of Allen and Pepe (1965) and Fischman (1967) and the biochemical techniques of Heywood and coworkers (1967, 1968a, 1968b, 1968c, 1969).

When consideration is given to the large number and variety of constituents and reactions involved in protein synthesis, it becomes very obvious why the techniques for protein biosynthesis are numerous and sophisticated (Figure 1). Techniques become even more complex when a study is made of the protein biosynthetic machinery of eucaryotes as opposed to procaryotes. The three major features observed in Figure 1 are replication, and . Replication or exact duplication of DNA is essential because DNA contains the genetic information for the synthesis of specific protein molecules. DNA polymerase is the responsible for 284. the catalysis of the exact replication of DNA. Transcription describez the events in which mRNA takes the genetic information from DNA in the nucleus and carries it to the in the . The combinatim of rrXNA and ribosomes in the cytoplasm constitute the elements upon which the pAroce;s of protein synthesis takes place. Transcription is catalyzed by the enzyne, RNA polymerase. The translational process involves the arrival of mino acids in the form of activated amino acyl-t-RNA, the sequential assembly of these mino acids, and the formation of bonds. Eventually the formation of a protein molecule will take place. Obviously, then, the study of a system of this kind requires a number of preparative and analytical techniques. Table 1 contains a list of preparative techniques essential for a study of muscle protein biosynthesis. Quantitative analyses refers to those techniques required to principally determine nitrogen, protein, and nucleic acids. Munro and Fleck (1966c,b; 1969) have presented excellent reviews of these techniques. Isolation media and homogenization are neces- sary for proper sample collection. These techniques vary according to investigators but the techniques of Heywood et d. (1967) have been success- fully employed to prepare highly active polysomes.-- Differential and density gradient centrifugation are indispensable tools for isolating and fraction- ating ribosomes (Heywood --et al., 1967). Tissue culture represents an excellent technique for the study of --in vitro muscle differentiation, development, and protein synthesis. Holtzer and his coworkers (Bischoff and Holtzer, 1970; Nameroff and Holtzer, 1969; Okajaki and Holtzer, 1965; 1966) have successfully employed these techniques. Recently, Reporter (1969) used tissue culture to study conver- sion of to 3 methylhistidine and its turnover rate in actin and myosin.

Analytical techniques are enumerated in Table 2. Spectrophotometry is very useful in determining the kind and quantity of nucleic acids (Heywood --et ELL., 1967). Electrophoresis has been very useful in the identification of the synthesized products, particularly myosin (Heywood et a.l., 1967). It has also been useful in the study of ribosomal proteins7LG --et al., 1969; Spiegel --et al., 1970). Chromatography has been useful in identification (Heywood and Rich, 1968) and purification (Baril and Hermann, 1967) of myosin. Kabat and Rich (1969) have successfully used autoradiography to show that muscle fibers are the major site of muscle synthesis. Fluorescence microscopy has provided information about myogenesis (Okajaki and Holtzer, 1965; Coleman and Coleman, 1968). Electron microscopy of muscle differentia- tion and development have been elegantly done by Allen and Pepe (1965) and Fischman (1967) . Message (1968) has also investigated muscle development using electron microscopy. Enzyme assays are vaLuable techniques because they provide evidence for the appearance or existance of certain components during differentiation and development. AMP-deaminase (Kendrick-Jones and Perry, 1967), ATPase (Trayer and Perry, 1966; Obinato, 1969), DNA polymerase (O'Neill and Strohman, 1969), creatine (Coleman and Coleman, 1968) , RNA polymerase (Marchok and Wolff, 1968; Breuer and Florini, 1966; Florini and Brewer, 1966) and thymidylate kinase (Scholl et al., 1968) are examples of associated with muscle development. The technique of isotopic incorporation is essential in determining the ability of ribo- somes to synthesize proteins (Heywood --et al., 1967). Now I would like to discuss in somewhat more detail the structural and biochemical evidence for myofibrillogenesis. Allen and Pepe (1965), with the electron microscope, were able to show that thin filaments (actin) appeared before thick f ilanent s (myosin) in developing chick embryo muscle cells (Figure 2). The thin filaments appeared in stage 16 (Figure Za) and by stage 18 large numbers of wavy thin filaments appeared (Figure 2b) and also thin filaments were observed interspersed with thick filaments (Figure Zc). Thick filaments were only observed in aggregates of thin and thick filaments. Also at this stage the first appearance of large poly- ribosomes were observed. In stage 20 (Figure 3) an increase of thin and thick filaments into myofibrils occurred. After four days of development, stage 24 myotond cells represent every stage of development (Figure 4) . The typical banding pattern of mature myofibrils are observed at stage 28 (Figure 5). Figure 6 shows the polyribosome structure containing 70-75 ribosomes thought to be associated with thick filaments (myosin) synthesis. A number of questions still remain unanswered about the appearance and relationship of certain constituents and particularly about the time of morphological appearance of thin and thick filaments. Fischman (1967), although disagreeing with Allen and Pepe (1965) about the time of appear- ance of thin filaments, also has done some very interesting work on chick embryo muscle cell differentiation and development with the electron microscope. Figure 7 shows a cross section through regions of three adjacent developing muscle fibers and their constituents. To be noted is the hexagonal array of thick and thin filaments in the myofibril and the free thick and thin filaments in the cytoplasm. A longitudinal section of this same material shows the various stages of myofibril formation (Figure 8). Free actin and myosin filmnts as well as two different stages of myofibril formation can be clearly observed. The first stage (Mfl) contains thick and thin filamnts, but without visible Z lines, and a later stage (Mfz) contains 2, 1, and A bands. Thin filaments were always observed to be in a 7 to 10 fold excess of thick filaments. Fischman (1967) concluded that thin filaments did not appear before thick filaments, although all of his work was done on 12-day chick embryo muscle. Figure 9 shows a fully formed myofibril with characteristic adult morphology.

Now I would like to direct your attention to the biochemical information on skeletal muscle protein biosynthesis. This work has emanated basically from four laboratories in this country. Brewer and Florini (1964, 1965, 1966), Florini and Brewer (1965, 1966) and Florini (1970) were early contributors and they have made a number of studies dealing with some of the hormonal factors influencing protein synthesis. Wool and coworkers (Castles and Wool, 1970; Kurihara and Wool, 1968; Low --et d., 1969; Martin and Wool, 1968; Wool and Cavicchi, 1966; 1967; and Wool and Kurihara, 1967) have made an intensive investigation of the effect of on protein biosynthesis. Young (Young and Alexis, 1968; Young -cet al., 1968; md Young and Hyang, 1969) have been interested in the effect of diet and . Heywood and coworkers (Heywood --et al., 1967; 1968; Heywood and Nwagwu, 1968; 1969; Heywood and Rich, 1968; and Heywood, 1970) have done the most comprehensive study of the synthesis of myofibrillar proteins. I would like to very briefly describe some of Heywood's experiments and their results on the biosynthesis of myofibrillar proteins. These experiments are also an excellent example of how biochemical techniques can complement electron microscopy techniques. It was thought that the large polyribosomes of muscle mediated myosin synthesis, but this had not been elucidated biochemically until Heywood --et al. (1967) recently gave dimension to mRNA coding for myosin. Polyribosomes were prepared from embryonic chick muscle as shown in Figure 10 (Heywood --et al., 1967). They also showed that the ionic strength of the 286. extracting medium was very important in obtaining ribosomes (Figure lla) . A buffered isolation medium consisting of 0.25 M KC1 resulted in larger yields of ribosomes by preventing the coprecipitation of ribosomes and polysomes with myosin. Heywood --et al., (1968) subsequently studied this problem in more detail. Also, the radioactivity counts were found to be associated entirely with polTyribosomes (Figure llb). To locate the polysomes active in myosin synthesis, the polysome sucrose gradient was divided into four fractions (Figure 12). A had the largest polysome class, B was next, then C, and finally D, which was composed largely of monomers and dimers (75s). The system for studying -~in vitro protein synthesis of these four fractions is shown in Table 3. The characteristics of the system are presented in Table 4 (Heywood --et al., 1967). The protein synthesized by the polyribosomes of fraction A, consisting of 50-60 ribosomes was identified as myosin by acrylamide gel electrophoresis (Figure 13). From the size of the polysomal cluster, it was also inferred that the polypeptide' chain being synthesized had a molecular weight of 170,000-200,000, this being about the size of the myosin subunits.

Larson _.-et al., (1969) have demonstrated with electron microscopy that a consistent and orderly relationship exists between polyribosomes and myosin filaments. Subsequent experiments by Heywood and Rich (1968) demon- strated that not only was fraction A identified as the polysomes synthesizing myosin, but that fraction B, containing 15-25 ribosomes, was involved with actin synthesis; fraction C, containing 5-9 ribosomes, was associated with tropomyosin synthesis; and fraction D, consisting of mostly monomers, was not involved in protein synthesis. Acrylamide gel electrophoresis was used to identify the specific protein synthesized (Figure 14). Fraction A again showed one major peak with the same mobility as myosin. Fraction B had two or three major peaks with the slowest peak migrating with the same mobility as actin. Fraction C had two major peaks with the most rapid peak having the same mobility as tropomyosin. It was inferred from these experiments that myosin, actin and tropomyosin were synthesized on monocistronic mRNAs. It was also observed (Figure 15) that the polysomal pattern varied as a function of the developmental age of the embryo. In the ten day embryo there is a peak in the polysome region associated with actin synthesis. The appearance of actin synthesizing polysomes before myosin synthesizing polysomes substantiates Allen and Pepe's (1965) observation that thin filaments appea before thick filaments. By fourteen days there has been a significant increase in myosin synthesizing polysomes and very little change in actin synthesizing polysomes. At this time there is also an increase in polysomes in the region of tropomyosin synthesis. These biochemical results agree with the electron microscope studies in that myosin and actin filaments are associated in a hexagonal array before the characteristic Z band is observed. Earlier evidence indicated that the Z line was composed of tropomyosin, but recent evidence indicates that this may not be the protein composing the Z line.

A subsequent study by Heywood and Nwagm (1968) found that the RNA extracted from large polysomes contained a single population of mRNA specifi- cally directing the --de novo synthesis of a protein electrophoretically and chromatographically identified as myosin. Myosin was found in chromatographic fractions 26-28 (Figure 16). Further investigations (Heywood and Nwagwu, 1969) showed that the 263 ribonucleic acid from fraction A was presumptive mRNA for the large 200,000 molecular weight subunit of myosin by 1) causing the forma- tion of polysomes in a cell-free system (Figure 17);Z) synthesizing a protein which both precipitated with antimyosin and cochromatographed with myosin on DEAE-cellulose (Table 5 and Figure 18); and 3) specifically synthesizing the 287. large, major component of myosin distinguishable by acrylamide gel electro- phoresis (Figure 19). Recently, Heywood (1970) has shown that in eucaryotes as in procaryctes, that mechanism for the initiation of protein synthesis involving ribosomal subunits is similar.

It has been shown that many of the same techniques that have given us information about the biochemistry and structure of muscle have also given us information about muscle protein biosynthesis. Yost of the work on protein biosynthesis up until recently, has been accomplished with procaryotes and reticulocytes as the source of protein synthesizing constituents. Now we know considerably more about muscle protein biosynthesis in eucaryotes; however, little is known about how and why certain meat animals grow more rapidly and efficiently than others, and how their growth is regulated. Utilization and adaptation of techniques should help us understand the nature and regulation of cellular and molecular components involved in myofibrillar protein synthesis. Therefore, a study of protein biosynthesis of meat animals is necessary because: 1) myofibrillar proteins (actin, myosin, tropomyosin) alone constitute 50-5576 of the cellular protein. Hence, they are the largest contributor to growth and development, of muscle. Consequently, feed efficiency, rate of gain, and efficient production of meat is largely dependent on the growth and regulation of myofibrillar proteins. Of the large quantities of feedstuffs, many of which could be used directly for human food consumption, ingested by meat animals, only a fraction is used for the growth and develop- ment of edible protein for human consumption. A primary reason for this inefficient process is because little is known about regulation of muscle protein biosynthesis, and of course many nutrients are used for body main- tenance and the synthesis of other non-edible constituents. 2) myofibrillar proteins are responsible for muscle contraction and movement in the living animal and for rigor mortis post-mortem; and 3) myofibrillar proteins as a food have a high biological value because they contain the essential amino acids for human health. Hence, increased quantities of muscle proteins are needed to meet world food problems. The use of techniques to study protein biosynthesis and regulation should result in both economical and humanitaim benefits .

Literature Cited

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2. Baril, E. F., and H. Hermann. 1967. Studies of muscle development. 11. Immunological and enzymatic properties and accumulation of chromatograph- ically homogeneous myosin of the leg musculature of the developing chick. Develop. Biol. 15:318. 3. Bischoff, R., and H. Holtzer. 1970. Inhibition of myoblast fusion after one round of DNA synthesis in 5-bromodeoxyuridine. J. Cell Biol. 44:134. 4. Breuer, C. B., M. C. Davies, and J. R. Florini. 1964. Amino acid incorporation into protein by cell-free preparations from rat skeletal muscle. 11. Preparation ard properties of muscle ribosomes and poly- ribosomes. Biochemistry 3:1713. 288.

5. Breuer, C . B., and J. R. Florini. 1965. Amino acid incorporation into protein by cell free systems from rat skeletal muscle. IV. Effects of animals age, androgens, and anabolic agents on activity of muscle ribo- somes. Biochemistry 4, 1544. 6. Breuer, C. B., and J. R. Florini. 1966. Effects of ammonium sulfate, growth , and propionate on ribonucleic acid poly- merase and chromatin activities in rat skeletd muscle. Biochemistry 5, 3857.

7. Castles, J. J., and I. G. Wool. 1970. Polyuridylic acid directed binding of phenylalanyl transfer ribonucleic acid to mammalian 40s ribosomal subunits. Biochemistry 9, 1909.

8. Chen, S. C., and V. R. Young. 1968. Preparation and some properties of rat skeletal muscle polyribosomes. Biochem. J. 106, 61.

9. Coleman, J. R., and A. W. Coleman. 1968. Muscle differentiation and macromolecular synthesis. J. Cell Physiol. 72, Sup. 1, 19.

10. Fischman, D. A. 1967. An electron microscope study of myofibril formation in embryonic chick skeletal muscle. J. Cell Biol. 32,

11. Florini, J. R., and C. B. Breuer. 1965. Amino acid incorporation into protein by cell-free preparations from rat skeletal muscle. 111. Comparisons of activity of muscle and ribosomes. Biochemistry 4, 253.

12. Florini, J. R., and C. B. Breuer. 1966. Amino acid incorporation into protein by cell free systems from rat skeletal muscle. V. Effects of pituitary on activity of ribosomes and ribonucleic acid polymerase in hypophysectomized rats. Biochemistry 6, 1870.

13. Florini, J. R. 1970. Effects of testosterone on qualitative pattern of protein synthesis in skeletal muscle. Biochemistry 9, 909.

14. Heywood, S. M. 1970. Formation of the initiation complex using muscle messenger . Nature 225, 696.

15. Heywood, S. M., R. M. Dowben, and A. Rich. 1967. The identification of polyribosomes synthesizing myosin. Proc. Natl. Acad. Sci. U. S. 57, 1002.

16. Heywocd, S. M., R. M. Dowben, and A. Rich. 1968. A study of muscle polyribosomes and the coprecipitation of polyribosones with myosin. Biochemistry 7: 3289.

17. Heywood, S. M., and M. Nwagwu. 1968. --De novo synthesis of myosin in a cell-free system. "roc. Natl. Acad. Sci. U. S. 60, 229.

18. Heywood, S. M., and M. Nwagwu. 1969. Partial characterization of pre- sumptive myosin messenger ribonucleic acid. Biochemistry 8, 3839.

19. Heywood, S. M., and A. Rich. 1968. --In vitro synthesis of native myosin, actin, and tropomyosin from embryonic chick polyribosomes. Proc. Natl. Acad. Sci. U. S. 59, 590. 289.

20. Ingram, V. M. 1966. The Biosynthesis of . New York. W. A. Benjamin, Inc.

21. Kabat, D., and A. Rich. 1969. The ribosomal subunit poliribosome c,clt in protein synthesis of embryonic skeletal muscle. Bioc iemistr, 6, 3742.

22. Kendrick-Jones, J., and S. V. Perry. 1967. The enzpes of adenlne in developing skeletal muscle. Biochem. J. 103, 2C7. 23. Kwihara, K., and I. G. Wool. 1968. Effect of insulir, on the synthesis of sarcoplasmic and ribosomal proteins of muscle. Nature 219, 721.

24. Larson, P. F., P. Hudgson, and J. N. Walton. 1969. Morphological relationship of polyribosomes and myosin f ilanents in developing and regenerating skeletal muscle. Nature 222, 1168.

25. Law, R. B., I. G. Wool, and T. E. Martin. 1969. Skeletal muscle ribosomal proteins. General characteristics and effect of diabetes. Biochim. Biophys. Acta. 194, 190.

26. Marchok, A. C., and J. A. Wolff. 1968. Studies of muscle development. IV. Some characteristics of RNA polymerase activity in isolated nuclei from developing chick muscle. Biochim. Biophys. Acta 155, 378.

27. Martin, T. E., and I. G. Wool. 1968. Formation of active hybrids from subunits of muscle ribosomes from normal and diabetic rats. Proc. Natl. Acad. Sei. 60, 569.

28. Message, M. A. 1968. Some problems associated with the development of the mammalian skeletal muscle fiber. In "Growth and Development of Mammals", (G. A. Lodge and G. E. Lamming, ea.) Plenum Press, New York, N. Y., p. 26.

29. Munro, H. N. 1969. A general survey of techniques used in studying in whole animals and intact cells. In "Mammalian Protein Metabolism", Vol. 111, p. 237 (H. N. Munro, ed.rAcademic Press, New York, N. Y.

30. Munro, H. N., and A. Fleck. 1966a. Recent developments in the measure- ment of nucleic acids in biological materials. Analyst 91, 78.

31. Munro, H. N., and A. Fleck. 196613. The determination of nucleic acids. -In "Methods of Biochemical Analysis", (D. Glick, ed.) Interscience and Wylie, New York, Vol. 1.4, p. 113.

32. Munro, H. N., and A. Fleck. 1969. Analysis of tissues and body fluids for nitrogenous constituents. In "Mammalian Protein Metabolism", Vol. 111, p. 423 (H. N. Munro, ed.) Acadezc Press, New York, N. Y.

33. Nameroff, M., and H. Holtzer, 1969. Contact-mediated reversible suppres- sion of myogenesis. Develop. Biol. 19, 380.

34. Obinata, T. 1969. The myosin of developing chick embr:;o. Arch. Bixhern. Biophys. 132, 184. 290.

35. Okazaki, K., and H. Holtzer. 1965. An analysis of myogenesis using fluorescein-labeled antimyosin. J. Histochem. Cytochem. 13, 726.

36. Okazaki, K., and H. Holtzer. 1966. Myogenesis: Fusion, myosin synthesis, and the mitotic cycle. Proc. Natl. Acad. Sei. U. S. 56, 1484.

37. O’Neill, M., and R. C. Strohman. 1969. Changes in DNA polymerase activity associated with cell fusion in cultures of embryonic muscle. J. Cell Physiol. 73, 61.

38. Reporter, M. 1969. 3-methylhistidine metabolism in proteins from cultured mammalian muscle cells. Biochemistry 8, 3489.

39. Scholl, A., H. Hermann, and J. S. Roth. 1968. Studies of muscle develop- ment. 111. An evaluation of thymidylate kinase activity with respect to cell proliferation in developing chick leg muscle. Sciences 7, 91.

40. Spiegel, M., E. S. Spiegel, and P. S. Meltzer. 1970. Qualitative changes in the basic protein fraction of developing embryos. Develop. Biol. 16, 474.

41. Strohman, R. C., E. W. Cerwinsky, and D. W. Holmes. 1964. Protein synthesis in developing muscle: Incorporation of Ik- into protein by muscle minces. Exptl. Cell Res. 35, 617.

42. Trayer, I. P., and S. V. Perry. 1966. The myosin of developing skeletal muscle. Biochemische Zeitschrift 345, 87.

43. Wool, I. G., and P. Cavicchi. 1966. Insulin regulation of protein synthesis by muscle ribosomes: Effect of the hormone on translation of messenger RNA for a regulatory protein. Proc. Natl. Acad. Sci. 56, 991.

44. Wool, I. G., and P. Cavicchi. 1967. Protein synthesis by skeletal muscle ribosomes. Effect of diabetes and insulin. Biochemistry 6, 1231.

45. Wool, I. G., and K. Kurihara. 1967. Determination of the number of active muscle ribosomes: Effect of diabetes and insulin. Proc. Natl. Acad. Sci. U. S. 58, 2401.

46. Young, V. R., and S. D. Alexis. 1968. In vitro activity of ribosomes and RNA content of skeletal muscle in young rats fed adequate or low protein. J. Nutr. 98, 255.

47. Young, V. R., S. C. Chen, and J. MacDonald. 1968. The sedimentation of rat skeletal muscle ribosomes. Biochem. J. 106, 913.

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Table 1. Preparative Techniques for the Study of Skeletal Muscle Protein Biosynthesis.

Technique Reference

Quantitative analyse s Munro and Fleck (1966a, b; 1969)

Isolation media Heywood --et al. (1967)

Homogenization Heywood --et al. (1967) Differential and density gradient centr if qation Heywood --et al. (1967) Tissue culture Okazaki and Holtzer (1966) Bischoff and Holtzer (1970)

Table 2. Analytical Techniques for the Study of Skeletal Muscle Protein Bi9synthesis.

T e chni que Reference

Spectrophotometry Heywood --et al. (1967) Electrophoresis Heywood --et al . ( 1967) Chromatography Heywood and Rich (1968) Microscopy Aut or ad i ogr aphy Kabat and Rich (1969) Fluorescence Okazaki and Holtzer (1965) Electron Allen and Pepe (1965), Fischman (1967) Enzyme assays AMP-de aminase Kendrick-Jones and Perry (1967) A!TP-ase Trayer and Perry (1966) DNA-polymerase O'Neill and Strohman (1969) RNA- polyme ra se Marchok and Wolff (1968) Thymidyl ate kinase Scholl --et al. (1968) Amino acid incorporation Heywood --et al. (1967) 292.

Table 3. Requirements for --In Vitro Amino Acid Incorporation. Salts (0.15 M KC1, 0.01 M MgC12)

Buffer (0.01 M Tris, pH 7.4)

Reducing agent (6 mM 2 mercaptoethanol)

Energy (2 mM ATP and 0.5 mM GTP)

Energy generation (10 mM phosphoenolpyruvate and 50 lug pyruvate kinase)

Amino acids (5 pM each of 20 amino acids containing 0.5 uc of CI4 labeled amino acid) t -RNA

Polyribosomes

pH 5.0 enzynes (transfer proteins)

Table 4. Characteristics of Amino Acid Incorporation in Polysomal Cell-Free System from Embryonic Chick Muscle (Taken from Heywood --et al., 1967).

Incubat ion lnedium Counts per minute Total counts (%)

C omplet e 32,200 100 -ATP, PEP, pyruvate kinase 80 0.3

-GTP 5,200 16.2

-pH 5 enzyme fraction 6,200 19.4 -Polyribosomes 2,000 6.2

Complete + 50 pg ribonuclease 60 0.2 293.

Table 5. Antimyosin-Precipitable Radioactivity (cpm) a (Taken from Heywood and Nwagwu, 1969)

Polysomal RNA Ant imyo s in Fraction (S) Total Released Precipitable

A, 28-32 400 150 74 A, 25-27 780 310 185 A, 17-19 370 160 50 A, 10-12 450 140 32 B, 25-27 36 0 155 57 B, 10-12 850 270 48 None 375 150 52 a The polysome RNA fractions are denoted by their approxi- mate sedimentation coefficient. (A) RNA from A polysomes; (B) RNA from B polysomes. Released radioactivity was taken as the radioactivity not sedimenting at 150,000 g; 20 ug of carrier myosin was added to the supernatants of the incubation mixtures before antimyosin was added. The antigen-antibody precipitate was washed in buffer and pre- cipitated with hot trichloroacetic acid. 294.

ltxact replication] DNA polymerase

/ RNA polymerase

i a.a.AMP-enz ATP + a a. - Specific enzyme a.a.-a a.-a.3.-a.a. .. a.a.-t-RNA

Specific enzyme Genetic control Peptide chain t-UNA of primary structure +------9 \’ I Folded peptide chain I I I <‘arhohydrate ------Prosthetic groups Protein subunit

Assembly of protein subunits

Figure 1. The overall scheme of DNA, RNA and protein synthesis. (T&en from Ingram, 1966). 295.

Figure 2. Electron micrograph of stage 16 chick embryonic muscle (2a). Section stained with PTA. Note scanty amount of filamentous material (arrows) . 40, OOOX. Electron micrograph of stage 18 (2b) . Note the high concentration of thin filaments. No thick filaments are seen. Section stained with PTA. Polyribosome (P) . 50,OOOX. Electron micrograph of filament aggregates in stage 18 (2c). Many thin fila- ments are seen interspersed with thick filaments. Tubules may be forming between aggregates (arrows) . Section stained with PTA. 144,OOOX. (Taken from Allen and Pepe, 1965). 296.

Figure 3. Electron micrograph of stage 20 of chick embryonic muscle. Note linear aggregation of thick and thin filaments. Polyribosomes (arrows) are seen in the cytoplasm. (Nucleus (N); golgi (Go). 40,OOOX. (Taken from Allen and Pepe, 1965.) 297.

Figure 4. Micrograph of a longitudinal section of stage 24 of chick embryonic muscle. Tubules (T) ; nucleus (N) ; golgi (Go) ; glycogen (Gl) ; polyribosome (P) ; cross-section of myofilaments (F) . 40,OOOX. (Taken from Allen and Pepe, 1965). 298

Figure 5. Micrograph of stage 28 chick embryonic muscle. Typical banding patterns (A, I, H, M, Z) are seen. The association between tubules (T) and Z line is apparrent. Polyribosome (P) . Unstained sections. 40,OOOX. (Taken from Allen and Pepe, 1965). 299.

Figure 6. Micrograph of polyribosom containing 70 to 75 ribosomes. 123,OOOX. (Taken from Allen and Pep, 1965). 300.

Figure 7. Electron micrograph of a cross-section through regions of three adjacent muscle fibers from a 12-day chick embryo. Sections me through thin and thick filmnt overlap (A-1) and the 2 band (2). Amorphous layer (AL) ; free myofilaments (Mf) ; nucleus (N) ; nuclear pores (NP). Glutaraldehyde-0s04 fixation; AraLdite embedding; FTA, UrAc, Pb citrate staining. 47,OOOX. (Taken from Fischman, 1967). 301.

Figure 8. Longitudinal section of previous micrograph. Stages of myofibril formation ranging from free myofilaments to complete myofibrils, are seen. Actin filaments (AI?) and myosin filaments (MF') are clearly distinguishable. Ribosome (R), (G) , and centrioles (C) are visible. Two stages of myofibrils formation are labeled Mi?1 and Mf2. The first stage (Mfl) contains thick and thin myofilaments in roughly parallel, longitudinal aggregation but without visible Z bands. A later developmental stage contain- ing Z, 1, and A bands is seen in mz. 50,OOOX. (Taken from Fischman, 1967). 302.

Figure 9. A fully formed myofibril with characteristic adult morphology is shown. Sarcomere bands (A, I, Z, and M) j plasma membranes (PM) ; nucleus (N) . 32,OOOX. (Taken from Fischman, 1967). 303.

Figure 10. Preparation of Skeletal Muscle Polyribosomes.

Dissection of embryonic leg muscle I I 0.25 M KC1, 0.01L M QCl, 0.01 M Tris, pH 7.4

HomogenizationJ

Layering of cytoplasmic extract on a 15- 40% linear sucrose gradient .L Centrifugation at 25,000 rpm for 2 hr

Collection of fractions and analysis at 260 mu I ..L --In vitro amino acid incorporation 304.

I I I I I I 2 .o

I .5 I i E 1.0 g .N- 0.6 > k zv, w 0 0.4 J a ul- 0a 0.2

I I I I I 0 5 IO 15 20 25 FRACTION NUMBER FRACTION NUMBER

(a) (6)

Figure 11. Sucrose gradient patterns of cytoplasmic extracts from embryonic chick muscle tissue. (a) Polysomal patterns obtained after extraction with buffers containing various concentrations of KC1 plus 0.01 M MgC12 and 0.01 M Tris (pH 7.4). Direction of centrifugation is to the left and the small arrow on the base line represents the last fraction. (b) Nascent protein radio- activity and optical density of polysomes isolated with 0.25 M KCI buffers. (Taken from Heywood et al., 1967). 305.

FRACTION NUMBER

Figure 12. Sucrose gradient analysis of cytoplasmic extract from 0.7 gm of 14 day old embryonic chick leg muscles. Fraction A through D were prepared for cell-free protein synthesis, while fractions in the cross hatched areas were not used. (Taken from Heywood and Rich, 1968). 306.

DISTANCE IN CM DISTANCE IN CM

Figure 13. Acrylamide gel electrophoresis of radioactive proteins from --in vitro incorporation of polysomes from fraction A-D. The solid line is a myosin marker. Two different periods of electrophoresis are shown. (Taken from Heywood -Let al., 1967). 307.

Figure 14. Distribution of radioactive proteins on acrylmide gel electro- phoresis. The electrode buffer was at pH 8.6. A 0.2 ml sample in 12 M urea, 0.1% 2-mercaptoethanol, and 50% sucrose was layered on the 12M urea gel (Taken from Heywocd acd Rich, 1968). 308.

I I 1 CHICK MUSCLE POLY SO MES

FRACTION NUMBER

Figure 15. Sucrose gradient analysis of cytoplasmic extracts from 0.7 gm of embryonic chick leg muscle at different ages of embryonic develop- ment (Taken from Heywood and Rich, 1968). 309.

7 I I I I I 1 1 I

8C

60

z a o 40

20

0 1 I I I 1 I I I I I 10 20 30 FRACTION NUMBER 40

Figure 16. DEAE-cellulose cochromatography of radioactive products of RNA directed synthesis. After the reaction mixtures had been cen- trifuged, the supernatants were dialyzed ffs 2 hr against 0.02 M KqP207(pH 8.5) containing an excess of C amino acids. Then, 100 ug of myosin was added to each sample. Chromatography was accomplished by eluting the soluble proteins with 120 ml 0.02 M KqP207 (pH 8.5). Myosin was then eluted with 0.36 M KC1, 0.02 M KO207 (pH 8.5). The elution of myosin was followed by absorb- ance at 280 mu (fractions 26-28). Finally, 5-ml fractions were collected and assayed for radioactivity. (Taken from Heywood and Nwagwu, 1968). 310.

r I I 1 I I 29-31 S

I

5 IO 15 20 25 5 IO 15 20 25 5 IO 15 20 25 FRACTION NUMBER

Figure 17. Polysome formation in cell-free system. The numbers associated with each figure represent the estimated range of the sedimenta- tion coefficients of the RNA fraction added to the cell-free system. The control is a cell-free system with no added RNA. (Taken from Heywood and Nwagwu, 1969). 311

I MYOS I N 400 -

25-27 S 300 -

f 0a 200- Ih.. 28-31 S

Figure 18. DEM-cellulose chromatography of radioactive products of RNA fractions from A polysomes. The elution of myosin was followed by absorbance at 280 npl (fractions 12-14). 5 ml fractions were col- lected and assayed for radioactivity. Numbers represent approxi- mate sedimentation coefficients of the different RNA fractions. The control has no RNA added to the reaction mixture. (Taken from Heywood and Nwagwu, 1969). 312.

I I I I I 1 4.0

n

82 3.0 Y* k v) 2.c az W V P

0a Fz 1.0 Q 0

DISTANCE IN CM

Figure 19. Acrylamide gel electrophoresis of fraction 12 from DEAE-cellulose chromatography of reaction mixtures (see Figure 8). A 0.2 ml sample was layered on each gel and run for 2 hr at 4 mA-tube. Direction of migration is to the right. A myosin marker was run in parallel. This gel was stained with Naphthol Blue Black and scanned at 500 w on a Gilford spectrophotometer gel scanner. The solid line denotes the position of the myosin marker; (- -) radio- activity of control, no RNA added; ( ****) radioactivity when 25-279 RNA from A polysomes was added to reaction mixture. The gels were sectioned into 1 mM slices after freezing. Radioactivity was measured by placing two adjacent slices on the same planchet and counting with a low-background counter. (Taken from Heywood and Nwagwu, 1969).