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. ============== Abstracts ============== 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. ----------------------------------------------------------------------------- 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. ----------------------------------------------------------------------------- [End Dicty News, volume 17, number 6]