Myosin is a family of proteins that demonstrates force-generating ATPase activity when it interacts with actin. Whereas myosins from vertebrate muscle and nonmuscle cells share common subunit composition and native structure, sarcomeric and nonsarcomeric myosin heavy chains (MHCs) consist of a distinct subfamily of isoforms. Individual MHC isoforms are encoded by different genes, and expression of various MHC genes is tightly associated with cell growth and differentiation. To study the mechanism for the MHC isoform switch during skeletal muscle differentiation, we have been using a mouse myogenic cell line (C2) as a model. Proliferating C2 myoblasts express nonmuscle MHCs abundantly, similar to NIH 3T3 fibroblasts. When C2 cells differentiate to form multinucleated myotubes, the nonmuscle MHC expression is dramatically decreased. Concomitantly, the sarcomeric MHC begins to be expressed. We have been searching for cis-regulatory element(s) which may be involved in upregulating nonmuscle MHC gene expression in C2 myoblasts and fibroblasts and/or downregulating it in C2 myotubes. Approximately 60 kb of human nonmuscle MHC-A gene, which contains 20 kb of 5' flanking region, exons 1 and 2 and 37 kb of intron 1, was segmented by restriction enzymes and introduced into the reporter gene construct which contains the nonmuscle MHC-A gene promoter and luciferase cDNA. Following transfection of various luciferase constructs, two fragments from the 1st intron, 0.5 kb and 2.8 kb in size, were found to cause a 3~10-fold increase in transcriptional activity in NIH 3T3 fibroblasts and C2 myoblasts, but not in the differentiated C2 myotubes. These will be further characterized to identify cis-acting elements. A previous study in this laboratory demonstrated the existence of neuron- specific isoforms of nonmuscle MHC-B, which may be generated by alternative splicing of pre-mRNA. To prove and study neuron-specific alternative splicing mechanism(s), we have been isolating the human genomic clones which encode this region of nonmuscle MHC-B. The neuron- specific 30 nt sequence was found to be a single exon and this exon was followed by the unconventional intron donor sequence, GCAAGT. In an attempt to localize the critical region of pre-mRNA for regulated alternative splicing, we have been constructing the minigene with deletion and/or mutation using a eukaryotic expression vector. Analysis of the mRNA derived from the minigene constructs, following transfection of the constructs into various cultured cells, is in progress.
