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dictyNews Volume 17 Number 06
Dicty News
Electronic Edition
Volume 17, number 6
Sept. 22, 2001
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu.
Back issues of Dicty-News, the Dicty Reference database and other useful
information is available at DictyBase--http://dictybase.org.
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Abstracts
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Regulated Expression of Myosin II Heavy Chain and RacB Using an Inducible
tRNA Suppressor Gene
Ka Ming Pang1,3 Theodore Dingermann2, and David A. Knecht1*.
1 Department of Molecular and Cell Biology, University of Connecticut
Storrs, Connecticut 06269 USA.
2.Institut fur Pharmazeutische Biologie Johann Wolfgang Goethe
Universitat Frankfurt, Germany.
Accepted: Gene
Abstract
An inducible expression system that indirectly regulates gene expression
through the use of an inducible suppressor tRNA has been used to express
both endogenous and exogenous genes in Dictyostelium. The tetracycline
repressor and tRNA suppressor (Glu) are expressed from a single G418
selectable vector, while a gene engineered to contain a stop codon is
expressed from a separate hygromycin selectable vector. b-galactosidase
could be induced over 300 fold with this system, and the extent of induction
could be varied depending upon the amount of tetracycline added. It took 3
days to fully induce expression, and about 3 days for expression to decrease
to baseline after removal of the tetracycline. Dictyostelium myosin II
heavy chain could also be expressed in an inducible manner, although the
induction ratio was not as high as b-galactosidase and the maximum
expression level was not as high as wild-type levels. A significant
accumulation of the truncated peptide indicates that complete suppression
of the stop codon was not achieved. Partial phenotypic reversion was
observed in null mutants inducibly expressing myosin II. RacB could also
be inducibly expressed, whereas the protein could not be expressed from a
constitutive promoter, presumably because expression at high levels is
lethal. Therefore, the inducible tRNA system can be used to control
expression of endogenous Dictyostelium genes.
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Altered Cell-Type Proportioning in Dictyostelium Lacking Adenosine
Monophosphate Deaminase
Soo-Cheon Chae, Danny Fuller and William F. Loomis*
Center for Molecular Genetics, Division of Biology
University of California San Diego, La Jolla, CA 92093
accepted: Developmental Biology
Abstract
The proportions of prespore and prestalk cells in Dictyostelium discoideum
are regulated such that they are size invariant and can adjust when the ratio
is perturbed. We have found that disruption of a gene, amdA, that encodes
AMP deaminase results in a significantly increased proportion of prestalk
cells. Strains lacking AMP deaminase form short, thick stalks and glassy
sori with less than 5% the normal number of spores. The levels of prestalk-
specific mRNAs in amdA- cells are more than twice as high as in wild type
strains and prespore specific mRNAs are reduced. Using an ecmA::lacZ
construct to mark prestalk cells, we found that amdA- null slugs have twice
the normal number of prestalk cells. The number of cells expressing an
ecmO::lacZ construct was not affected by loss of AmdA indicating that the
mutation results in an increase in PST-A prestalk cells rather than PST-O
cells. This alteration in cell-type proportioning is a cell autonomous
consequence of the loss of AMP deaminase since mutant cells developed
together with wild type cells still produced excess prestalk cells and
wild type cells carrying the ecmA::lacZ construct formed normal numbers
of prestalk cells when developed together with an equal number of
amdA- mutant cells.
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[End Dicty News, volume 17, number 6]
