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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]