Eukaryotic cells contain unique forms of myosin which may play a role in cell proliferation. Referred to as 'nonmuscle' myosins, these proteins share a greater degree of amino acid identity 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 cells and the expression of these proteins in lymphocytes infected with retroviruses. Previous investigators have isolated two forms of mammalian nonmuscle myosin heavy chain (NMMHC) cDNA. These isoforms, from chicken and human cDNA libraries, have been designated """"""""A"""""""" and """"""""B"""""""". Using restriction enzyme digestion of a previously sequenced cDNA B clone, a probe has been generated near the 3' portion of the clone. A human T-cell (Jurkat) library was screened with this probe resulting in the isolation of new clones. This allowed for extension of NMMHC B sequence in the 3' direction. A second probe generated from this clone is currently being used to screen another T-cell library. NMMHC A and B oligonucleotide probes have been synthesized in order to distinguish between the two isoforms. Southern analysis reveals that these A and B differential probes can selectively discriminate between the two isoforms. Human genomic DNA and Northern blots of human T-cell RNA are currently being probed. RNA from HTLV-I-infected T-cells has been prepared for Northern analysis with these differential probes. HTLV-I-infected cells have been chosen because retroviral genes appear to integrate into the host's generic material upon infection, altering normal gene expression. If successful, this experiment may provide information about mRNA expression in cells responding to infection by retroviruses.
