All eukaryotic cells contain one or more isozymes of nonmuscle myosin. Two forms of the myosin heavy chain (MHC-A and MHC-B) have been cloned in this laboratory and are the products of two different genes in humans. One of the isozymes, MHC-B, is also subject to alternative splicing of messenger RNA to yield four possible variants (due to the existence of combinations of two possible inserted nucleotide sequences in the head region of myosin). We would like to understand the function of the MHC-A and MHC-B isoforms and the function of the insertions in the head region of the MHC-B isoform. To do this, we wish to over-express the myosin head and the rod fragments from human and frog (Xenopus laevis) in cell lines of the respective species. Over-expression of the rod fragment, in particular, may contribute to a loss of function because, although the rod can form filaments with intact myosin, it lacks the enzymatic and actin binding sites located in the head. The human myosin fragments will be subcloned into a vector for packaging in a retrovirus. The melanoma cell line, A2058, will be infected with virus and studied for phenotypic changes. Since this cell line has previously been used for studies of cell motility, it will be possible to measure the response of infected and control cells (cells infected with vector alone or mock-infected cells). Other cell lines will be either infected with retrovirus containing myosin or transfected with plasmid constructs by lipofection or electroporation. We have recently isolated the cDNA encoding the MHC-A isoform from Xenopus laevis. These clones are now being sequenced. The rod and head clones, along with those for the MHC-B isoform, will be used for transfections into the frog cell lines, A6 and XTC. Antisense constructs will also be used in the same vectors in an effort to interrupt myosin function.
