Evidence for inserted sequences in the head region of nonmuscle myosin specific to the nervous system. Cloning of the cDNA encoding the myosin heavy chain-B isoform of vertebrate nonmuscle myosin

Abstract

The complete amino acid sequence of a vertebrate nonmuscle myosin heavy chain-B isoform (MHC-B, 1976 amino acids, 229 kDa) has been deduced by using cDNA clones from chicken brain libraries. The chicken nonmuscle MHC-B shows overall similarity in primary structure to other MHCs in the areas contributing to the ATP-binding site and actin-binding site. Similar to other nonsarcomeric MHC IIs, there is a short uncoiled tail sequence at the carboxyl terminus of the molecule. It is in the uncoiled tail sequence that the greatest number of differences in amino acids sequence between MHC-A and B were found, which allowed generation of isoform-specific antibodies. These antibodies were used to determine the relative content of MHC-A and MHC-B in various tissues. During the cloning of the cDNA encoding chicken brain MHC-B, we found a 63-nucleotide insertion encoding 21 amino acids located in the head region of the MHC near to the actin-binding site and a 30 nucleotide insertion encoding 10 amino acids near to the ATP-binding site. Analysis using S-1 nuclease showed that both inserts are expressed in a tissue-dependent manner; mRNA containing the inserts is present in tissues of the nervous system, but is absent from other non-muscle cells, which contain the noninserted isoform of MHC-B. Similar inserts were found in corresponding positions in human cerebellar mRNA. Antibodies raised against a peptide synthesized based on the 21 amino acid insert found in chickens recognize a MHC isoform in the same tissues that are enriched for the mRNA. These insertions appear to be a mechanism for generating additional MHC-B isoforms specific to the nervous system.