Molecular genetic characterization of nonmuscle myosin heavy chain

Abstract

The goal of this project involves the isolation and characterization of a human nonmuscle myosin heavy chain (MHC) isoform and the analysis of the interaction between sarcomeric myosin heavy chain and nonmuscle myosins when co-expressed in the same cell. A 5.2 Kb cDNA clone encoding part of a human nonmuscle MHC isoform was isolated and completely sequenced. This gene utilizes at least two polyadenylation signals which do not appear to vary in different cell types. Thee is a small number of nonmuscle MHC genes and the gene encoding the MHC isolated is located in human chromosome 22. In order to identify the sequences responsible for nonmuscle filament assembly, a segment from the rod encoding the last 203 amino acids of the nonmuscle MHC cDNA clone isolated was expressed in E. coli. This fragment encode the sequences needed for filament assembly as assessed by its solubility profile at different ionic strengths and it forms paracrystals with 43 and 14 nm periodicities. Transient transfection of cardiac {dollar}\alpha{dollar} MHC into three different cell types was used to study the ability of cardiac MHC to interact with the endogenous cytoskeleton. The pattern of expression of cardiac MHC depends upon the cell environment. The pattern of expression of sarcomeric MHC was also analyzed in CCl{dollar}\sb4{dollar}-induced cirrhotic fat storing cells. At least one of the cells clones isolated, "naturally" express adult and perinatal sarcomeric MHC and their protein is distributed throughout the cytoplasm in apparently different compartment than the filamentous actomyosin stress fibers structure of the cell.