Cloning of Contractile Protein Genes

Schwartz, Robert J.; Stone, Edwin M

doi:10.1007/978-1-4615-9296-9_7

Cited by 10 publications

(4 citation statements)

References 228 publications

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“…The switching of isotypic mRNA expression may result from selective transcription or repression of members ofa multigene family (26) or from differences in RNA processing (5) or stability. Such control mechanisms are hypothesized to underlie developmental changes in myoblast populations in vertebrate embryos (38). It was initially thought that gene amplification might also play a role in control of contractile gene expression (51).…”

Section: Discussionmentioning

confidence: 99%

Sequential expression of chicken actin genes during myogenesis.

Hayward¹,

Schwartz²

1986

The Journal of Cell Biology

164

128

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Abstract. Embryonic muscle development permits the study of contractile protein gene regulation during cellular differentiation. To distinguish the appearance of particular actin mRNAs during chicken myogenesis, we have constructed DNA probes from the transcribed 3' noncoding region of the single-copy a-skeletal, a-cardiac, and S-cytoplasmic actin genes. Hybridization experiments showed that at day 10 in ovo (stage 36), embryonic hindlimbs contain low levels of actin mRNA, predominantly consisting of the a-cardiac and B-actin isotypes. However, by day 17 in ovo (stage 43), the amount of a-skeletal actin mRNA/#g total RNA increased more than 30-fold and represented -90% of the assayed actin mRNA. Concomitantly, a-cardiac and fl-actin mRNAs decreased by 30% and 70%, respectively, from the levels observed at day 10. In primary myoblast cultures, fl-actin mRNA increased sharply during the proliferative phase before fusion and steadily declined thereafter, aCardiac actin mRNA increased to levels 15-fold greater than a-skeletal actin mRNA in prefusion myoblasts (36 h), and remained at elevated levels. In contrast, the a-skeletal actin mRNA remained low until fusion had begun (48 h), increased 25-fold over the prefusion level by the completion of fusion, and then decreased at later times in culture. Thus, the sequential accumulation of sarcomeric a-actin mRNAs in culture mimics some of the events observed in embryonic limb development. However, maintenance of high levels of a-cardiac actin mRNA as well as the transient accumulation of appreciable a-skeletal actin mRNA suggests that myoblast cultures lack one or more essential components for phenotypic maturation.TINS are essential components for many forms of cellular organization and mobility. Actin proteins have been well characterized in warm-blooded vertebrates, where at least seven different actins comprise a multigene family that is expressed in both a temporal-and tissuespecific manner (2,9,10,16,18,33,37,45). In particular, the switching of actin gene expression has been studied during in vitro myogenesis. Our laboratory (37) as well as others (1,28,34) have shown that the nonmuscle B-and 3,-cytoplasmic actin mRNAs are the predominant actin mRNA species in cultured replicating myoblasts. During subsequent myoblast fusion and formation of multinucleated myotubes, the nonmuscle actin mRNAs are reduced in content. Concomitantly, the sarcomeric a-actin mRNAs are induced and attain relatively high levels within myotubes cultured in the absence of neurons.As members of a multigene family, the sarcomeric a-actin isoforms are encoded by the two closely related a-skeletal and a-cardiac genes (10). Comparison of the a-skeletal and acardiac amino acid sequences reveals only four amino acid substitutions within 375 residues, among the highest conservation found in vertebrate actins (46). Vandekerckhove and Weber (47) have shown that primitive chordates were the first eukaryotes to express a striated muscle-specific form. During the evolution of primitive amphibia or stem r...

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Section: Discussionmentioning

confidence: 99%

Sequential expression of chicken actin genes during myogenesis.

Hayward¹,

Schwartz²

1986

The Journal of Cell Biology

164

128

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show abstract

“…On the other hand, levels of a-skeletal actin mRNA were not significant until 76 h when >700 copies of probe hybridized per myotube nucleus. In situ hybridization efficiency was estimated at ~10% of the a-striated actin mRNA molecules (15,000 copies) previously detected per 75-h myotube nucleus (30).…”

Section: Calculation Of the Number Of Probe Molecules Hybridized Per Differentiated Cell And The Ejsiciency Of In Situ Hybridizationmentioning

confidence: 99%

Cellular localization of muscle and nonmuscle actin mRNAs in chicken primary myogenic cultures: the induction of alpha-skeletal actin mRNA is regulated independently of alpha-cardiac actin gene expression.

Hayward

Zhu

Schwartz

1988

The Journal of Cell Biology

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Abstract. Specific DNA fragments complementary to the 3' untranslated regions of the 13-, ~t-cardiac, and ~t-skeletal actin mRNAs were used as in situ hybridization probes to examine differential expression and distribution of these mRNAs in primary myogenic cultures. We demonstrated that prefusion bipolar-shaped cells derived from day 3 dissociated embryonic somites were equivalent to myoblasts derived from embryonic day 11-12 pectoral tissue with respect to the expression of the ~t-cardiac actin gene. Fibroblasts present in primary muscle cultures were not labeled by the ~t-cardiac actin gene probe. Since virtually all of the bipolar cells express a-cardiac actin mRNA before fusion, we suggest that the bipolar phenotype may distinguish a committed myogenic cell type. In contrast, a-skeletal actin mRNA accumulates only in multinucleated myotubes and appears to be regulated independently from the ct-cardiac actin gene. Accumulation of a-skeletal but not ~t-cardiac actin mRNA can be blocked by growth in Ca2+-deficient medium which arrests myoblast fusion. Thus, the sequential appearance of a-cardiac and then ~t-skeletal actin mRNA may result from factors that arise during terminal differentiation. Finally, the 13-actin mRNA was located in both fibroblasts and myoblasts but diminished in content during myoblast fusion and was absent from differentiated myotubes. It appears that in primary myogenic cultures, an asynchronous stage-dependent induction of two different a-striated actin mRNA species occurs concomitant with the deinduction of the nonmuscle fi-actin gene.

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“…Differentiation of muscle cells involves the developmental progression of mesodermal cells into committed proliferating myoblasts, which subsequently withdraw from the cell cycle and fuse to form myotubes (41). Myoblast fusion is accompanied by the repression of a subset of nonmuscle genes and the broad activation of a family of muscle-specific genes (10,40).…”

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confidence: 99%

Activation of skeletal alpha-actin gene transcription: the cooperative formation of serum response factor-binding complexes over positive cis-acting promoter serum response elements displaces a negative-acting nuclear factor enriched in replicating myoblasts and nonmyogenic cells.

Lee¹,

Chow²,

Fang³

et al. 1991

Mol. Cell. Biol.

142

103

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Three upstream CBAR cis-acting promoter elements, containing the inner core CC(A/T)6GG of the serum response element (SRE), are required for myogenic cell type-restricted expression of the avian skeletal alpha-actin gene (K.L. Chow and R.J. Schwartz, Mol. Cell. Biol. 10:528-538, 1990). These actin SRE elements display differential binding properties with two distinct nuclear proteins, serum response factor (SRF) and another factor described here as F-ACT1. SRF is able to bind to all actin SREs with various affinities. This multisite interaction is marked by cooperative binding events in that the two high-affinity proximal and distal SREs facilitate the weak central-site interaction with SRF, leading to the formation of a higher-order SRF-promoter complex. Functional analyses reveal that undisrupted multiple SRF-DNA interactions are absolutely essential for promoter activity in myogenic cells. F-ACT1, present at higher levels in nonmyogenic cells and replicating myoblasts than in myotubes, binds solely to the proximal SRE, and its binding is mutually exclusive with that of SRF owing to their overlapping base contacts. The cooperative promoter binding by SRF, however, can effectively displace prebound F-ACT1. In addition, an intact F-ACT1 binding site acts as a negative promoter element by restricting developmentally timed expression in myoblasts. F-ACT1 may therefore act as a repressor of skeletal alpha-actin gene transcription. This interplay between F-ACT1 and SRF may constitute a developmental as well as a physiologically regulated mechanism which modulates sarcomeric actin gene expression.

show abstract

Cloning of Contractile Protein Genes

Cited by 10 publications

References 228 publications

Sequential expression of chicken actin genes during myogenesis.

Sequential expression of chicken actin genes during myogenesis.

Cellular localization of muscle and nonmuscle actin mRNAs in chicken primary myogenic cultures: the induction of alpha-skeletal actin mRNA is regulated independently of alpha-cardiac actin gene expression.

Activation of skeletal alpha-actin gene transcription: the cooperative formation of serum response factor-binding complexes over positive cis-acting promoter serum response elements displaces a negative-acting nuclear factor enriched in replicating myoblasts and nonmyogenic cells.

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