Refined conditions for selective modifications of rabbit skeletal myosin light chains

Biochimie ◽  
1992 ◽  
Vol 74 (12) ◽  
pp. 1083-1090 ◽  
Author(s):  
J.M. Burgat ◽  
A. Roulet ◽  
R. Cardinaud
Keyword(s):  
Light Chains ◽  
Myosin Light Chains ◽  
Skeletal Myosin

scholarly journals Synthesis of rat myosin light chains in heterokaryons formed between undifferentiated rat myoblasts and chick skeletal myocytes.

1981 ◽  
Vol 91 (1) ◽  
pp. 11-16 ◽  
Author(s):  
W E Wright
Keyword(s):  
Primary Cultures ◽  
Muscle Cells ◽  
Thigh Muscle ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Control Of Gene Expression ◽  
Cell Functions ◽  
Cell Extracts ◽  
Skeletal Myosin ◽  
Species Specific

The control of gene expression during terminal myogenesis was explored in heterokaryons between differentiated and undifferentiated myogenic cells by analyzing the formation of species specific myosin light chains of chick and rat skeletal muscle. Dividing L6 rat myoblasts served as the biochemically undifferentiated parent. The differentiated parental cells were mononucleated muscle cells (myocytes) that were obtained from primary cultures of embryonic chick thigh muscle by blocking myotube formation with EGTA and later incubating the postimitotic cells in cytochalasin B. Heterokaryons were isolated by the selective rescue of fusion products between cells previously treated with lethal doses of different cell poisons. 95-99% pure populations of heterokaryons formed between undifferentiated rat myoblasts and differentiated chick myocytes were obtained. The cells were labeled with [35S]methionine, and whole cell extracts were analyzed on two-dimensional polyacrylamide gels. These heterokaryons synthesize the light chain of chick myosin and both embryonic and adult light chains of rat skeletal myosin. Control homokaryons formed by fusing undifferentiated cells to themselves did not synthesize skeletal myosin light chains. Control heterokaryons formed between undifferentiated rat myoblasts and chick fibroblasts also failed to synthesize myosin light chains. These results indicate that differentiated chick muscle cells provide some factor that induces L6 myoblasts to synthesize rat myosin light chains. This system provides a model for investigating the processes by which differentiated cell functions are induced.

scholarly journals Induction of muscle genes in neural cells.

1984 ◽  
Vol 98 (2) ◽  
pp. 427-435 ◽  
Author(s):  
W E Wright
Keyword(s):  
Cell Lines ◽  
Neural Cell ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Neural Cells ◽  
Cell Hybrids ◽  
Skeletal Myosin ◽  
Muscle Genes ◽  
Inducing Factors ◽  
Neural Cell Lines

The regulation of skeletal muscle genes was examined in heterokaryons formed by fusing differentiated chick skeletal myocytes to four different rat neural cell lines. Highly enriched populations of heterokaryons isolated using irreversible biochemical inhibitors were labeled with [35S]methionine and analyzed on two-dimensional gels. Rat skeletal myosin light chains were induced in three of the four cell combinations. The one exception, the S-20 cholinergic cell line, not only failed to synthesize rat muscle proteins but also suppressed chick myogenic functions. Experiments with heterokaryons between chick myocytes and cells from whole embryonic rat brain cultures demonstrated that rat skeletal myosin light chains are inducible in normal diploid neural cells as well as in established neural cell lines. In contrast, dividing cell hybrids between rat myoblasts and rat glial cells were nonmyogenic. These results demonstrate that although neural cells may contain factors that prevent the decision to differentiate along myogenic lines in cell hybrids, most neural cell lines do not dominantly suppress the expression of muscle structural genes in heterokaryons. Furthermore, the skeletal myosin light chain genes in most neural cell lines are regulated by a mechanism that permits them to respond to putative chick skeletal myocyte-inducing factors. The "open" state of these myogenic genes may explain many of the reports of apparent "transdifferentiation" to muscle in neural cultures and neural tumors.

Dictyostelium discoideum myosin: isolation and characterization of cDNAs encoding the regulatory light chain

1989 ◽  
Vol 9 (7) ◽  
pp. 3073-3080
Author(s):  
S R Tafuri ◽  
A M Rushforth ◽  
E R Kuczmarski ◽  
R L Chisholm
Keyword(s):  
Amino Acid ◽  
Light Chain ◽  
Base Pair ◽  
Calcium Binding ◽  
Expression Patterns ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Base Pairs ◽  
Isolation And Characterization ◽  
Skeletal Myosin

Phosphorylation of the regulatory light chains (RMLC) of nonmuscle myosin can increase the actin-activated ATPase activity and filament formation. Little is known about these regulatory mechanisms and how the RMLC are involved in ATP hydrolysis. To better characterize the nonmuscle RMLC, we isolated cDNAs encoding the Dictyostelium RMLC. Using an antibody specific for the RMLC, we screened a lambda gt11 expression library and obtained a 200-base-pair clone that encoded a portion of the RMLC. The remainder of the sequence was obtained from two clones identified by DNA hybridization, using the 200-base-pair cDNA. The composite RMLC cDNA was 645 nucleotides long. It contained 60 base pairs of 5' untranslated, 483 bases of coding, and 102 base pairs of 3' untranslated sequence. The amino acid sequence predicted an 18,300-dalton protein that shares 42% amino acid identity with Dictyostelium calmodulin and 30% identity with the chicken skeletal myosin RMLC. This sequence contained three regions that were similar to the E-F hand calcium-binding domains found in calmodulin, troponin C, and other myosin light chains. A sequence similar to the phosphorylation sequence found in chicken gizzard and skeletal myosin light chains was found at the amino terminus. Genomic Southern blot analysis suggested that the Dictyostelium genome contains a single gene encoding the RMLC. Analysis of RMLC expression patterns during Dictyostelium development indicated that accumulation of this mRNA increases just before aggregation and again during culmination. This pattern is similar to that obtained for the Dictyostelium essential myosin light chain and suggests that expression of the two light chains is coordinated during development.

scholarly journals Dictyostelium discoideum myosin: isolation and characterization of cDNAs encoding the regulatory light chain.

1989 ◽  
Vol 9 (7) ◽  
pp. 3073-3080 ◽  
Author(s):  
S R Tafuri ◽  
A M Rushforth ◽  
E R Kuczmarski ◽  
R L Chisholm
Keyword(s):  
Amino Acid ◽  
Light Chain ◽  
Base Pair ◽  
Calcium Binding ◽  
Expression Patterns ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Base Pairs ◽  
Isolation And Characterization ◽  
Skeletal Myosin

Phosphorylation of the regulatory light chains (RMLC) of nonmuscle myosin can increase the actin-activated ATPase activity and filament formation. Little is known about these regulatory mechanisms and how the RMLC are involved in ATP hydrolysis. To better characterize the nonmuscle RMLC, we isolated cDNAs encoding the Dictyostelium RMLC. Using an antibody specific for the RMLC, we screened a lambda gt11 expression library and obtained a 200-base-pair clone that encoded a portion of the RMLC. The remainder of the sequence was obtained from two clones identified by DNA hybridization, using the 200-base-pair cDNA. The composite RMLC cDNA was 645 nucleotides long. It contained 60 base pairs of 5' untranslated, 483 bases of coding, and 102 base pairs of 3' untranslated sequence. The amino acid sequence predicted an 18,300-dalton protein that shares 42% amino acid identity with Dictyostelium calmodulin and 30% identity with the chicken skeletal myosin RMLC. This sequence contained three regions that were similar to the E-F hand calcium-binding domains found in calmodulin, troponin C, and other myosin light chains. A sequence similar to the phosphorylation sequence found in chicken gizzard and skeletal myosin light chains was found at the amino terminus. Genomic Southern blot analysis suggested that the Dictyostelium genome contains a single gene encoding the RMLC. Analysis of RMLC expression patterns during Dictyostelium development indicated that accumulation of this mRNA increases just before aggregation and again during culmination. This pattern is similar to that obtained for the Dictyostelium essential myosin light chain and suggests that expression of the two light chains is coordinated during development.

scholarly journals Hybrids of Physarum myosin light chains and desensitized scallop myofibrils.

1981 ◽  
Vol 90 (2) ◽  
pp. 408-414 ◽  
Author(s):  
V T Nachmias
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Atpase ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Free Calcium ◽  
Standard Methods ◽  
Regulatory Light Chains ◽  
Skeletal Myosin

The two light chains of Physarum myosin have been purified in a 1:1 ratio with a yield of 0.5-1 mg/100 g of plasmodium and a purity of 40-70%; the major contaminant is a 42,000-dalton protein. The 17,700 Mr Physarum myosin light chain (PhLC1) binds to scallop myofibrils, providing the regulatory light chains (ScRLC) have been removed. The 16,500 Mr light (PhLC2) does not bind to scallop myofibrils. The calcium control of scallop myosin ATPase is lost by the removal of one of the two ScRLC's and restored equally well by the binding of either PhLC1 or rabbit skeletal myosin light chains. When both ScRLC's are removed, replacement by two plasmodial light chains does not restore calcium control as platelet or scallop light chains do. Purified plasmodial actomyosin does not bind calcium in 10(-6) M free calcium, 1 mM MgCl2. No tropomyosin was isolated from Physarum by standard methods. Because the Physarum myosin light chains can substitute only partially for light chains from myosin linked systems, because calcium does not bind to the actomyosin, and because tropomyosin is apparently absent, the regulation of plasmodial actomyosin by micromolar Ca++ may involve other mechanisms, possibly phosphorylation.

Interaction of skeletal myosin light chains with calcium ions

Biochemistry ◽  
1978 ◽  
Vol 17 (12) ◽  
pp. 2319-2325 ◽  
Author(s):  
M. N. Alexis ◽  
W. B. Gratzer
Keyword(s):  
Calcium Ions ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Skeletal Myosin
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