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dictyNews Volume 29 Number 04
dictyNews
Electronic Edition
Volume 29, number 4
August 3, 2007
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu
or by using the form at
http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit.
Back issues of dictyNews, the Dicty Reference database and other
useful information is available at dictyBase - http://dictybase.org.
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Abstracts
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Using Single loxP Sites to Enhance Homologous Recombination:
ts Mutants in Sec1 of Dictyostelium discoideum.
Mark S. Bretscher* and Margaret Clotworthy
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
*corresponding author.
PloS-ONE, in press.
Background
Dictyostelium discoideum amoebae are haploid and, as they share many
features with animal cells, should be an ideal creature for studying basic
processes such as cell locomotion. Isolation of mutants in this amoeba has
largely been limited to non-essential genes: nsfA Ñ the gene for
NEM-sensitive factor Ñ remains the only essential gene for which conditional
(ts) mutants exist. These ts mutants were generated by gene replacement using
a library of mutagenised nsfA containing a selectable marker: transformants
were then screened for temperature sensitivity. The success of this approach
depended on the high level of homologous recombination prevailing at this
locus: ~95% of selected clones were homologous recombinants. This is
unusually high for Dictyostelium: homologous recombination at other loci is
usually much less, usually between 0-30%, making the isolation of ts mutants
much more tedious.
Methodology/Principal Findings
In trying to make ts mutants in sec1A, homologous recombination was found to
be only ~25%. A new approach, involving single loxP sites, was investigated.
LoxP sites are 34 bp sequences recognised by Cre recombinase and between
which this enzyme catalyses recombination. A Dictyostelium line containing
a single loxP site adjacent to the 3Õ end of the sec1A gene was engineered.
A sec1A replacement DNA also containing a single loxP site in a homologous
position was then introduced into this cell line. In the presence of CRE
recombinase, homologous recombination increased to ~80% at this locus,
presumably largely driven by intermolecular recombination between the two
single loxP sites.
Conclusions/Significance
A route to increase the rate of homologous recombination at a specific locus,
sec1A, is described which enabled the isolation of 30 ts mutants in sec1A.
One of these, sec1Ats1,has been studied and found to cease moving at the
restrictive temperature. The approach described here may be valuable for
enhancing homologous recombination at specified loci and thus for introducing
mutations into specific genes in Dictyostelium and other creatures.
Submitted by: Mark Bretscher [msb@mrc-lmb.cam.ac.uk]
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Immune-like Phagocyte Activity in the Social Amoeba.
Guokai Chen, Olga Zhuchenko and Adam Kuspa
Verna and Marrs McLean Department of Biochemistry and Molecular Biology,
Baylor College of Medicine, One Baylor Plaza, Houston TX 77030.
Science, in press
Social amoebae feed on bacteria in the soil, but they aggregate when starved
and form a migrating slug that will undergo fruiting body morphogenesis to
produce terminally differentiated spores and stalk cells. We describe a
previously unknown cell type in the social amoeba which appears to provide
detoxification and immune-like functions, which we term Sentinel (S) cells.
S cells were observed to engulf bacteria and sequester toxins while
circulating within the slug, eventually being sloughed off. A
Toll/Interleukin-1 Receptor (TIR) domain protein, TirA, was also required
for some S cell functions and for vegetative amoebae to feed on live bacteria.
This apparent innate immune function in social amoebae, and the use of TirA
for bacterial feeding, suggests an ancient cellular foraging mechanism that
may have been adapted to defense functions well before the diversification of
the animals.
Submitted by: Adam Kuspa [akuspa@bcm.tmc.edu]
--------------------------------------------------------------------------------
A cell-number counting factor regulates levels of a novel protein, SslA, as
part of a group-size regulation mechanism in Dictyostelium
Tong Gao, Celine Roisin-Bouffay, R. Diane Hatton, Lei Tang, Debra A. Brock,
Tiffany DeShazo, Laura Olson, Wan-Pyo Hong, Wonhee Jang, Elvia Canseco,
Deenadayalan Bakthavatsalam, and Richard H Gomer
Howard Hughes Medical Institute and Department of Biochemistry and Cell Biology,
Rice University, Houston, TX 77005-1892
Eukaryotic Cell, in press
Developing Dictyostelium cells form aggregation streams that break into groups
of ~2 x 104 cells. The breakup and subsequent group size are regulated by a
secreted multisubunit counting factor (CF). To elucidate how CF regulates
group size, we isolated second site suppressors of smlAø, a transformant that
forms small groups due to oversecretion of CF. smlAø/sslA1(CR11) cells form
roughly wild-type size groups due to an insertion in the beginning of the
coding region of sslA1, one of two highly similar genes encoding a novel protein.
The insertion increases levels of SslA. In wild-type cells, the sslA1(CR11)
mutation forms abnormally large groups. Reducing SslA levels by antisense causes
the formation of smaller groups. The sslA(CR11) mutation does not affect the
extracellular accumulation of CF activity or the CF components countin and CF50,
suggesting that SslA does not regulate CF secretion. However, CF represses
levels of SslA. Wild-type cells starved in the presence of smlAø cells,
recombinant countin, or recombinant CF50 form smaller groups, whereas sslA1(CR11)
cells appear to be insensitive to the presence of smlAø cells, countin, or CF50,
suggesting that the sslA1(CR11) insertion affects CF signal transduction. We
previously found that CF reduces intracellular glucose levels. sslA(CR11) does
not significantly affect glucose levels, while glucose increases SslA levels.
Together, the data suggest that SslA is a novel protein involved in part of a
signal transduction pathway regulating group size.
Submitted by: Richard Gomer [richard@rice.edu]
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[End dictyNews, volume 29, number 4]
