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Two clonal rat rhabdomyosarcoma cell lines BA-Han-1B and BA-Han-1C with different capacities for myogenic differentiation have been examined for the expression of muscle regulatory basic helix-loop-helix (bHLH) proteins of the MyoD family. Whereas cells of the BA-Han-1C subpopulation constitutively expressed MyoD1 and could be induced to differentiate with retinoic acid (RA), BA-Han-1B cells did not express any of the myogenic control factors and appeared to be largely differentiation-defective. Upon induction with RA, BA-Han-1C cells expressed also myogenin, in contrast to BA-Han-1B cells which never activated any of the genes encoding muscle bHLH factors. The onset of myogenin transcription in BA-Han-1C cells required de novo protein synthesis and DNA replication suggesting that RA probably did not act directly on the myogenin gene. Although MyoD1 was expressed in proliferating BA-Han-1C myoblasts, muscle-specific reporter genes were not activated indicating that MyoD was biologically inactive. However, transfections with plasmid expressing additional MyoD1 protein resulted in the transactivation of muscle genes even in the absence of RA. mRNA encoding the negative regulatory HLH protein Id was expressed in proliferating BA-Han-1C cells and disappeared later after RA induction which suggested that it may be involved in the regulation of MyoD1 activity. The myogenic differentiation of malignant rhabdomyosarcoma cells strictly correlated with the activation of the myogenin gene. In fact, stable transfections of BA-Han-1C cells with myogenin expressing plasmids resulted in spontaneous differentiation. Together, our results suggest that the transformed and undifferentiated phenotype of BA-Han-1C rhabdomyosarcoma cells is associated with the inactivation of the myogenic factor MyoD1 as well as lack of myogenin expression. RA alleviates the inhibition of myogenic differentiation, probably by activating MyoD protein and myogenin gene transcription. BA-Han-1B cells did not respond to RA and the differentiated phenotype could not be restored by overexpression of MyoD1 or myogenin.

MyoD is a member of a family of myogenic transcription factors with similar properties [1] including myogenin [2–4], Myf-5 [5], and Myf-6, also called MRF4 or herculin [6–8]. Transfection of the myogenic HLH genes into a wide range of cultured cells can induce the skeletal muscle differentiation program and activate muscle-specific genes. No substantial difference between the myogenic HLH genes has been revealed by these assays. The myogenic transcription factor genes are expressed solely in skeletal muscle, and thus, their role appears to be in determining the identity of the skeletal myocyte lineage [9–12].

The spatial and temporal expression pattern of the muscle regulatory gene Myf-6 (MRF4/herculin) has been investigated by in situ hybridization during embryonic and fetal mouse development. Here, we report that the Myf-6 gene shows a biphasic pattern of expression. Myf-6 transcripts are first detected in the most rostral somites of the mouse embryo at 9 d of gestation and accumulate progressively in myotomal cells along the rostro-caudal axis. This expression is transient and Myf-6 mRNA can no longer be detected in myotomal cells after day 12 post coitum (p.c.). In contrast to other muscle determination genes (MyoD1, myogenin, Myf-5), Myf-6 mRNA is not detected in limb buds or visceral arches and skeletal muscle of the mouse embryo (day 8-15 p.c.). In fetal mice, Myf-6 transcripts appear at day 16 p.c. in all skeletal muscles, and the gene continues to be expressed at a high level after birth. These results suggest that early Myf-6 expression may be restricted to a population of myogenic cells that does not contribute to the embryonic muscle masses in limb buds and visceral arches. The reappearance of Myf-6 mRNA in fetal skeletal muscle coincides approximately with secondary muscle fiber formation and the onset of innervation.

The muscle regulatory protein myogenin accumulates in differentiating muscle cells when the culture medium is depleted for serum. To investigate the regulation of myogenin gene expression, we have isolated and characterized the Myf4 gene which encodes the human homologue of murine myogenin. Serum components, basic FGF (b-FGF), transforming growth factor beta (TGF-beta), and EGF, agents which suppress differentiation of muscle cells in vitro, down-regulate the activity of the Myf4 gene, suggesting that it constitutes a nuclear target for the negative control exerted by these factors. The 5' upstream region containing the Myf4 promoter confers activity to a CAT reporter plasmid in C2C12 myotubes but not in fibroblasts and undifferentiated myoblasts. Unidirectional 5' deletions of the promoter sequence reveal that integral of 200 nucleotides upstream of the transcriptional start site are sufficient for cell type-specific expression. The forced expression of the muscle determining factors, MyoD1, Myf5, and Myf6 and to a lesser degree Myf4, results in the transactivation of the Myf4 promoter in C3H mouse 10T1/2 fibroblasts. Pathways potentially involved in conveying signals from the cell-surface receptors to the Myf4 gene were probed with pertussis- and cholera toxin, forskolin, and cAMP. Dibutyryl-cAMP and compounds that stimulate adenylate cyclase inhibit the endogenous Myf4 gene and the Myf4 promoter in CAT and LacZ reporter constructs. Conversely, pertussis toxin which modifies Gi protein stimulates Myf4 gene expression. In summary, our data provide evidence that the muscle-specific expression of the Myf4 gene is subject to negative control by serum components, growth factors and a cAMP-dependent intracellular mechanism. Positive control is exerted by a pertussis toxin-sensitive pathway that presumably involves G proteins.

The combination of DNA microinjection into tissue culture cells and the chloramphenicol acetyltransferase enzyme assay has been used to determine quantitatively the transcriptional activity of viral or eukaryotic promoters. As shown for a wide range of DNA concentrations and time periods between 2 and 16 h the assay allows the reliable and reproducible determination of promoter activities in defined cellular backgrounds. Only 50 cells are necessary for the test.

Human smooth muscle and nonmuscle cells express closely related myosin alkali light chains which are different from the isoforms present in striated muscle tissues. To dat no information of the amino acid sequence of these mammalian nonstriated muscle isoforms has been available. We have isolated full-length cDNA clones encoding the nonmuscle and smooth muscle myosin light chains from cultured human lymphoblasts and heart aorta smooth muscle cells, respectively. Here we present the complete nucleotide sequences for both cDNA clones, together with the deduced amino acid sequences for the peptides. Both cDNAs contain the same open reading frame for 151 amino acids with 5 amino acid differences located in the C terminus. These differences are encoded by a block of 44 nucleotides with is present only the smooth muscle mRNA. To identify the human gene coding for the two MLC isoforms, we have isolated and sequenced the nonmuscle MLC gene, together with several intronless pseudogenes. A singl e functional gene was found containing 7 exons which are utilized for the coding information of the SM MLC mRNA. In contrast, the NM MLC mRNA does not contain sequences encoded by exon 6 which corresponds to the 44 nucleotides sequences encoded by exon 6 which corresponds to the 44 nucleotides expressed in SM mRNA. This genomic configuration suggests that both the smooth muscle and nonmuscle MLCs in man are generated from the identical primary transcript by alternative splicing pathways taking place in a tissue-dependent manner.

In mammalian organisms the regulatory or phosphorylatable myosin light chains in heart and slow skeletal muscle have been shown to be identical and presumably constitute the product of a single gene. We analyzed the expression of the avian cardiac myosin light chain (MLC) 2-A in heart and slow skeletal muscle by a combination of experimental approaches, e.g., two-dimensional gel electrophoresis of the protein and hybridization of mRNA to specific MLC 2-A sequences cloned from chicken. The investigations have indicated that, unlike in mammals, in avian organisms the phosphorylatable myosin light chains from heart and slow skeletal muscle are distinct proteins and therefore products of different genes. The expression of MLC 2-A is restricted to the myocardium and no evidence was found that it is shared with slow skeletal muscle.

tRNA I Tyr and tRNA II Tyr have been purified from B.subtilis and their nucleotide sequence determined. tRNA I Tyr differs from tRNA II Tyr only by the extent of modification of the adenosine in 3′ position adjacent to the anticodon, i 6 A and ms 2 i 6 A respectively.