Eucaryotic cells contain unique forms of myosin which may play a role in cell proliferation and motility. Referred to as """"""""nonmuscle"""""""" myosins, these proteins share a greater degree of homology with smooth muscle myosin, but a lesser degree of similarity when compared to striated muscle myosin. Our interest is in the identification of nonmuscle myosins from human Jurkat T- lymphocytes and the expression of these proteins in other normal and abnormal cells. This laboratory has isolated two forms of mammalian nonmuscle myosin heavy chain (NMMHC) """"""""A"""""""" and """"""""B"""""""". Using restriction enzyme digestions of previously sequenced cDNA """"""""B"""""""" clones, we have created unique probes which have allowed us to isolate the entire translated region of NMMHC """"""""B"""""""" from a cDNA library. 1.5 kb of the 3' untranslated region and 200 nucleotides of the 5' untranslated sequence have also been identified. Sequencing the nucleotides of these cDNA clones has allowed us to predict the amino acid sequence and to create unique cDNA probes and antibodies specific for nonmuscle myosin isoforms. We have obtained human spleen, kidney, Kaposi's sarcoma, aorta, brain and spinal cord for evaluation. Human cell lines (A2058 melanoma, HeLa, HL-60 promyelocytes, and Jurkat) have also been evaluated. NMMHC """"""""A"""""""" and """"""""B"""""""" cDNA clones have been used as probes to distinguish between the two isoforms in different human tissues and cell lines. Northern blots were hybridized with radioactive cDNA probes and analyzed to evaluate the isoforms at the level of mRNA. Based on the nucleotide sequence, we have identified areas of low amino acid similarity between NMMHC """"""""A"""""""" and """"""""B"""""""" and have raised antibodies to these areas. An investigator in this laboratory (Masayuki Takahashi) has identified at least two tissue-specific nucleotide inserts coding for unique amino acids in chicken neural tissue. Utilizing PCR, these have been detected in human nervous tissues, allowing the generation of mammalian antibodies which should elucidate the inserts' distributions and locations on immunoblots and tissue sections.
