EXPRESSION OF NONMUSCLE MYOSIN ISOFORMS IN EUKARYOTIC CELLS

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Two forms of the nonmuscle myosin heavy chain (MHCIIA and MHCIIB) have been cloned in this laboratory and shown to be the products of two different genes. The isoforms are expressed in a tissue-dependent manner and, when present in the same cell, exhibit distinct localization. The MHCIIB isoform additionally shows alternative splicing in two areas of the MHC: one after amino acid 211, near the ATP-binding domain (PESPKPVKHQ in loop 1) and another near the actin- binding domain (loop 2). Biochemical characterization of the nonmuscle myosin IIB fragment, HMM which contains the sites of enzymatic activity and actin-binding has been carried out on HMM isolated from the Baculovirus expression system. There is no major difference in the ability of HMM with or without the inserted region in loop 1 to move actin filaments or to hydrolyze ATP. Smooth muscle MHC also shows alternative splicing in an identical area near the ATP-binding domain. Smooth muscle myosin containing this seven amino acid insert (QGPSFSY) moves actin filaments in the in vitro motility assay at a rate 2.5 times faster than the non-inserted isoform. HMM IIB has been engineered to contain the seven amino acid insert from smooth muscle by splicing the 21 nucleotides coding for the insert into the nonmuscle HMM cDNA. The ATPase activity of the HMM containing the insert from the smooth muscle MHC is two times higher than that of non- inserted HMM (0.268s-1 vs 0.128s-1). In the in vitro motility assay, the inserted HMM moves actin filaments at a five-times faster rate than the non-inserted HMM (0.275 +/- .061 vs 0.054 +/- .021). Current experiments, in collaboration with Lee Sweeney are underway to study the effect of mutations in the smooth muscle sequence on the enzymatic and mechanical activity of the inserted HMM. Of the two forms of nonmuscle myosin, one isoform, MHCII-B, has been targeted to generate a knockout mouse which does not express the protein encoded by the MHCII-B gene. The deletion of MHCII-B results in disruption of the cardiac myocytes and malformation of the heart as well as the brain. Phenotypic alterations in the heart resemble the human diseases tetralogy of Fallot and double outlet right ventricle. the goal of the current project is to delete the protein encoded by the MHCII-A gene, generating a knockout mouse for the MHCII-A. A genomic clone encompassing 7.5 kb of the MHCII-A gene and including exon 3 has been subcloned into the vector pPNT. In this construct, exon 3 is interrupted by the neomycin resistance gene which will be used for positive selection of embryonic stem cells. The thymidine kinase gene follows the MHCII-A gene fragment at the 3' end of the construct and will be used for negative selection in embryonic stem cells. In order to rule out effects of the neomycin resistance gene on the phenotype observed, the same genomic fragment, minus bp in exon 3, has been engineered into the lox P site-containing vector, pBS479 (gift of Brian Sauer, NIH). Upon mating a mouse carrying this integrated construct with a mouse expressing the Cre recombinase, a mouse which does not express MHCII-A and which does not contain the neomycin resistance gene will be generated.