From r-chisholm@nwu.edu Sat May 27 11:24 CDT 1995 Return-Path: Received: from dicty.cmsbio.nwu.edu by worms.cmsbio.nwu.edu (5.x/SMI-SVR4) id AA00888; Sat, 27 May 1995 11:24:49 -0500 Received: by dicty.cmsbio.nwu.edu with Microsoft Mail id <01BA2C24.A076E060@dicty.cmsbio.nwu.edu>; Sat, 27 May 1995 11:25:52 -0500 Message-Id: <01BA2C24.A076E060@dicty.cmsbio.nwu.edu> From: "Rex L. Chisholm" To: 'CSM-News' Subject: FW: CSM News, v4, #18 Date: Sat, 27 May 1995 11:25:50 -0500 Mime-Version: 1.0 Content-Transfer-Encoding: 7bit Content-Type: text/plain; charset="us-ascii" Content-Length: 5393 Status: RO ---------- From: csm-news@worms.cmsbio.nwu.edu Sent: Saturday,May 20,1995 2:30AM To: dd-email-list@worms.cmsbio.nwu.edu Subject: CSM News, v4, #18 CSM News Electronic Edition Volume 4, number 18 May 20, 1995 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmsbio.nwu.edu/dicty.html" =========== Abstracts =========== Cloning vectors for the production of proteins in Dictyostelium discoideum Dietmar J. Manstein, Hans-Peter Schuster, Piero Morandini$, and Deborah M. Hunt National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK and $ MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. Gene, in press. Summary We constructed and tested a series of cloning vectors designed to facilitate protein production and purification in Dictyostelium discoideum (Dd). These vectors carry the origin of replication of the Dd high copy number plasmid, Ddp2, expression cassettes consisting of the strong, constitutive actin (act) 15 or the inducible discoidin (dis) Ig promoters, a translational start codon upstream from a multiple cloning site, and sequences for the addition of epitope or affinity tags at the N- or C-termini of any protein. The affinity tag used corresponds to 7 (N-terminal fusion) or 8 (C-terminal fusion) histidine residues. The epitope tags correspond to an 11-amino-acid sequence from human c-myc, recognised by monoclonal antibody (mAb) 9E10, and the Glu-Glu-Phe sequence recognised by mAb YL1/2 to a-tubulin. Both these mAb are commercially available. The YL1/2 epitope offers a second affinity tag for the purification of proteins under native conditions. The functional competence of the vectors was tested by determining their ability to promote the expression of various Dd myosin constructs. High expression levels were obtained for each vector, up to 1 mg of homogenous, functional protein per g of cells was obtained after purification of the recombinant products. The cellular slime mold Dictyostelium discoideum (Dd) has many features that make it an attractive eukaryotic host for the production of recombinant proteins. Dd is biochemically well characterised and can be grown readily in the laboratory on a large scale. Our work is mostly focused on the characterisation of cytoskeletal proteins like myosin. Functional myosin motor fragments can be produced and purified in milligram quantities using Dd (Manstein et al., 1989) and detailed kinetic (Ritchie et al., 1993) and structural studies (Schroeder et al., 1993) have been carried out using myosin motor domains produced in Dd. The new vectors were designed to improve our ability to produce recombinant proteins in Dd for in vitro and in vivo studies. Introduction of the Glu-Glu-Phe epitope tag (Stammers et al., 1991) or poly-histidine (his) tag (Janknecht et al., 1991) facilitates the rapid and efficient purification of overexpressed protein and the Glu-Glu-Phe and c-myc epitope tags can be used for the analysis of protein function and fate within the cell. [Editors Note: The sequences of these vectors are available through the Dicty Web Page.] -------------------------------------------------------------------------- Expression of Light Meromyosin in Dictyostelium blocks normal myosin II function. C. Geoffrey Burns(1), Mary Reedy(1), John Heuser(2) and Arturo De Lozanne(1) (1) Department of Cell Biology, Duke University Medical Center Durham, NC 27710; (2)Department of Cell Biology, Washington University Saint Louis, MO 63110 J. Cell Biol., in press. Summary The ability of myosin II to form filaments is essential for its function in vivo. This property of self association is localized in the light meromyosin (LMM) region of the myosin II molecules. To explore this property in more detail within the context of living cells, we expressed the LMM portion of the Dictyostelium myosin II heavy chain gene in wild-type Dictyostelium cells. We found that the LMM protein was expressed at high levels and that it folded properly into a-helical coiled-coiled molecules. The expressed LMM formed large cytoplasmic inclusions composed of entangled short filaments surrounded by networks of long tubular structures. Importantly, these abnormal structures sequestered the cell's native myosin II, completely removing it from its normal cytoplasmic distribution. As a result the cells expressing LMM displayed a myosin-null phenotype: they failed to undergo cytokinesis and became multinucleate, failed to form caps after treatment with concanavalin A, and failed to complete their normal developmental cycle. Thus, expression of the LMM fragment in Dictyostelium completely abrogates myosin II function in vivo. The dominant-negative character of this phenotype holds promise as a general method to disrupt myosin II function in many cell types without the necessity of gene targeting. ------------------------------------------------------------------------ [End CSM News, volume 4, number 18]