Partial characterization of presumptive myosin messenger ribonucleic acid

Heywood, Stuart M.; Nwagwu, Mark

doi:10.1021/bi00837a050

Cited by 109 publications

(28 citation statements)

References 21 publications

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“…In particular the RNP containing the 26 S RNA, putative messenger for the large subunit of myosin [5], appears to be an important component in the post-transcriptional control of myosin gene expression [6]. This species, identified in the dividing myoblast and the fused myotube as a peak sedimenting at 26 S after pulse labelling of the cultures, undergoes an increase in stability just prior to fusion.…”

Section: Introductionmentioning

confidence: 99%

The use of metrizamide to separate cytoplasmic ribonucleoprotein particles in muscle cell cultures: A method for the isolation of messenger RNA, independent of its poly a content

Buckingham

Gros

1975

FEBS Letters

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Section: Introductionmentioning

confidence: 99%

The use of metrizamide to separate cytoplasmic ribonucleoprotein particles in muscle cell cultures: A method for the isolation of messenger RNA, independent of its poly a content

Buckingham

Gros

1975

FEBS Letters

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“…This RNA has been isolated from whole chick-embryo muscle and has been characterized in several earlier reports (17,18,22). We confirm, with the MHC mRNA from cultured cells, much of what has been found for the mRNA from whole muscle developing in vivo or in ovo.…”

Section: Discussionmentioning

confidence: 99%

Myosin Heavy Chain Messenger RNA from Myogenic Cell Cultures

Przybyla¹,

Strohman²

1974

Proc. Natl. Acad. Sci. U.S.A.

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The appearance of messenger RNA for myosin heavy chains in chick-embryo myogenic cell cultures was investigated. Total polyribosomes were isolated from cultures at various times of development and were purified in sucrose step gradients. These polysomes were either extracted with phenol or were treated with puromycin. The ribonucleoprotein particles and ribosomal subunits released by puromycin were fractionated on sucrose gradients. RNA from polysomes or from puromycin-dissociated subunits was fractionated on oligo(dT)-cellulose columns, and the bound and unbound RNA was assayed for activity of myosin heavy chain messenger RNA in a rabbit reticulocyte cell-free system. RNA stimulating myosin heavy-chain synthesis was found predominantly in the unbound fractions of the oligo-(dT)-cellulose columns. After puromycin treatment of polysomes, the myosin heavy chain messenger RNA, which sediments at 18-26 S, was associated with a ribonucleoprotein particle sedimenting between 30 and 40 S. Myosin heavy chain messenger RNA was obtained from cultures containing well-developed myotubes and from cultures undergoing myogenic cell fusion. This messenger RNA was not detectable in early, unfused cultures, or in later cultures in which myogernic cell fusion had been prevented by treatment with ethyleneglycol bis(fl-aminoethyl ether)-NN'-tetraacetic acid. These experiments demonstrate that messenger RNA for myosin heavy chains becomes associated with ribosomes only after myogenic cell fusion has begun.During the differentiation of the multinucleated, skeletal muscle fiber the time of myogenic cell fusion and the formation of the embryonic myotube is correlated with the time of intensive synthesis of myosin and the other muscle-specific enzymes (1-4). Presently at issue is whether cell fusion is actually coupled to the regulatory mechanisms that control this intensified protein synthesis. Evidence from this laboratory on fusion-arrested chick myogenic cell cultures strongly suggests that such a coupling mechanism exists (2), and a recent report from another laboratory (5) suggests that in ratmuscle cell cultures there is a post-transcriptional regulatory event that is correlated with cell fusion.In this paper we report on the isolation of an mRNA fraction derived from polysomes of cultured chick-embryo myogenic cells. This RNA directs the incorporation of radioactive leucine into myosin heavy chains (MHCs) in a cellfree system.

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“…Heywood and Nwagwu (1969) found that ribosOBies actively supported synthesis of myosin heavy chains when the ribosomes and myosin mRNA were both isolated from the same embryonic chicken muscle (i.e., in a homologous system). When this same assay was performed using a heterologous system (ribosomes isolated from chicken reticulocytes and myosin mRNA isolated from embryonic 5^ chicken muscle), however, only a very low level of myosin synthesis occurred (Heywood, 1969)• Hence, ribosomes from a nonmuscle source apparently were very inefficient relative to muscle ribosomes in supporting synthesis of myosin heavy chains directed by presumptive myosin mRNA.…”

Section: ^2mentioning

confidence: 99%

“…Rather than attempting indirect studies, such as those reported by Buckingham et al (197^), and in view of the uncertainties involving ability to quantitatively initiate protein synthesis on myosin mRNA's (Heywood and Nwagwu, 1969) or to assay purified myosin mRNA in a cellfree assay as attempted by Strohman and coworkers (Pryzbyla et al, 1973;Pryzbyla and Strohman, 197^;Strohman et al, 197'+), a more direct approach was taken. If prefusion muscle cells synthesize myosin, then these cells must con tain myosin polysomes.…”

Section: ^2mentioning

confidence: 99%

“…Because of the decrease in specific radioactivity of leucine and the resulting spurious decrease in myosin heavy-chain synthesis when large amounts of polysomes were added to the in vitro protein synthesis g^say used in this study, however, the amount of polysomes added to each assay was adjusted so that approximately 600 to 1,000 dpm's were incorporated into myosin heavy chains during the assay per iod; In this way it was possible to minimize, although not completely eliminate, errors in measuring nascent myo sin heavy-chain synthesis due to the observed nonlinearity in rate of myosin synthesis at different levels of added Figure 16, the endogenous control was about lOfo of the total dpm's incorporated into myosin heavy chains in the presence of polysomes (for com parison see Figure 7). This high rate of endogenous in corporation was evidently due to myosin mRNA adhering to the muscle ribosomes during preparation (Heywood and Nwagwu, 1969)• To decrease this high endogenous incorporation and to attempt simultaneously to stimulate initiation, the muscle ribosomes in these assays were replaced with rabbit reticulocyte ribosomes prepared as described in Materials and Methods. Reticulocytes synthesize very little myosin compared to embryonic muscle, and ribosomes isolated from reticulocytes, therefore, have very low endogenous incor poration into myosin.…”

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confidence: 99%

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Effect of myoblast fusion during muscle cell differentiation on myosin polysome accumulation and myosin synthesis

Young

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Partial characterization of presumptive myosin messenger ribonucleic acid

Cited by 109 publications

References 21 publications

The use of metrizamide to separate cytoplasmic ribonucleoprotein particles in muscle cell cultures: A method for the isolation of messenger RNA, independent of its poly a content

The use of metrizamide to separate cytoplasmic ribonucleoprotein particles in muscle cell cultures: A method for the isolation of messenger RNA, independent of its poly a content

Myosin Heavy Chain Messenger RNA from Myogenic Cell Cultures

Effect of myoblast fusion during muscle cell differentiation on myosin polysome accumulation and myosin synthesis

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