374 Biochimica et Biophysica Acta, 950 (1988) 374-384 Elsevier BBA 91837 Expression of the human cardiac aetin gene in differentiating rat skeletal myoblasts Martha M. Ruben, Michae!A. Rudnicki, Trevor S. Bladon, Karen Jardine, Jane Craig, and Michael W. McBurney Departments of Medicine and Biology, University of Ottawa, Ottawa C¢'anada) (Received 18 December 1987) (Revised manuscript received 28 April 1988) Key words: Myogenesis;Gene regulation; Cardiac actin gene; (Human gene); (Rat myoblast) The human cardiac-actin (Cn-actin) gene was transfected into rat L6 skeletal myoblasts and stable transformants were isolated. The level of the CH-actin transcript varied between clones but changed little during the differentiation of myoblasts into multinucleate myotubes. Chimeric genes were constructed in which the CH-actin promoter, first non-coding exon (44 bp), and first intron (about 700 bp) were linked to the Herpes simplex virus thymidine kinase (tk) coding region. Clones of L6 cells transformed with these chimeric genes contained variable levels of actin-tk mRNA which changed little during differentiation. Thus, the activity of the Cn-actin promoter appeared not to be up-regulated upon differentiation of myoblasts into myotubes. In clones of cells expressing the actin-tk mRNA, the TK protein was not detected in myoblasts but appeared in differentiating multinucleate myotubes. We interpret these results as suggesting develop- mentally regulated translation of the actin.tk mRNA. Since the first 44 nucleotides of the actin-tk mRNA were derived from the 5'-untranslated region of the Cn-actin mRNA. These experiments suggest that translation of the actin-tk mRNA may be controlled by this region. Introduction Multinucleate skeletal muscle develops from mononucleate proliferating myoblast cells which withdraw from the cell cycle, fuse to neighbouring myocytes, and express a variety of muscle-specific proteins. The initial appearance of many muscle- specific proteins is concomitant with the ap- pearance of their mRNAs [1], suggesting that muscle-specific gene expression is regulated prim- Abbreviations: CH-actin, human cardiac actin (gene); TK (tk), thymidine kinase (EC 2.7.1.21); EC, embryonal carcinoma; PBS, phosphate-buffered saline; nt, nucleotide. Correspondence: M.W. McBurney, Department of Medicine, University of Ottawa, 451 Smytli Road, Ottawa, Canada, K1H 8M5. arily at the level of mRNA accumulation [2], probably by transcriptional control [3]. To in- vestigate the nature of muscle gene regulation, a number of investigators have transfected skeletal muscle genes into skeletal myoblast cell lines and, in some cases, the level of mRNA from the trans- fected gene was elevated after differentiation of myoblasts into myotubes [4-6]. We have previously transfected the human cardiac actin (Cn-actin) gene into multipotential embryonal carcinoma (EC) cells and selected sta- ble transformants. The levels of CH-actin mRNA rose dramatically in cultures of these EC cells during their differentiation into cardiac muscle [7]. The cardiac actin gene is normally expressed in cardiac and embryonic skeletal muscle cells [8,9]. In order to investigate the expression of the C H- actin gene in cells differentiating along the skeletal 0167-4781/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)