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 Tlymphocytes 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. A substantial portion of the untranslated region has also been identified. The Polymerase Chain Reaction will be employed to obtain the remaining 3' untranslated region from T-cell cDNA. A primer extension will determine the extent of the 5' untranslated region. Sequencing the nucleotides of these cDNA clones has allowed us to predict the amino acid sequence and to create unique oligonucleotide probes and antibodies specific for nonmuscle myosin isoforms. Currently, we have obtained human spleen, kidney and brain for evaluation. NMMHC """"""""A"""""""" and """"""""B"""""""" oligonucleotide probes have been synthesized in order to distinguish between the two isoforms in these different human tissues. Northern blots will be hybridized with radioactive oligonucleotides 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 developed antibodies to these areas. After the distribution of nonmuscle myosins has been determined in normal tissues, we are interested in looking into disease states, such as retrovirally infected T-lymphocytes or Kaposi's Sarcoma, an HIV-related tumor.
