dictyNews Electronic Edition Volume 28, number 9 April 13, 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. ========= Abstracts ========= Ethylene induces zygote formation through an enhanced expression of zyg1 in Dictyostelium mucoroides Aiko Amagai, Sho-saku Soramoto, Shu-hei Saito and Yasuo Maeda Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aoba, Sendai 980-8578, Japan Exp Cell Res., in press We have previously demonstrated that a potent plant hormone, ethylene induces sexual development including zygote formation in Dictyostelium cells, and that a novel gene (zyg1) is also involved in zygote formation. Based on these findings, the present work was mainly designed to reveal 1) the precise relationship between the ethylene amount and zygote formation, and 2) the relation of in situ ethylene synthesis to zyg1 expression, using transformants that over- or under-produce ACC-oxidase (Dd-aco) involved in ethylene biosynthesis. ACOOE cells overexpressing Dd-aco gene overproduced ethylene and exhibited the augmented zygote formation. In contrast, ACO-RNAi cells, in which the expression of Dd-aco was suppressed by the RNAi method, showed a reduced level of ethylene production, thus resulting in inhibition of zygote formation. Importantly, the expression of zyg1 was affected by the amount of ethylene produced: Zyg1 expression was augmented in ACOOE cells, but was significantly suppressed in ACO-RNAi cells. In another experiment, we found that 1-methylcyclopropene (1-MCP), which is known to inhibit the function of ethylene by binding specifically to ethylene receptors, greatly suppresses zygote formation. These results indicate that ethylene is capable of inducing zygote formation through the expression of zyg1. Submitted by: Aiko Amagai [aiamagai@mail.tains.tohoku.ac.jp] -------------------------------------------------------------------------------- A G protein coupled receptor with a lipid kinase domain is involved in cell density sensing Deenadayalan Bakthavatsalam1+, Derrick Brazill2, Richard H. Gomer3, Ludwig Eichinger1, Francisco Rivero1* and Angelika A. Noegel1* 1 Center for Biochemistry, Institute for Biochemistry I, Medical Faculty and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany 2 Center for the Study of Gene Structure and Function, Hunter College, City University of New York, New York, USA 3 Howard Hughes Medical Institute and Department of Biochemistry and Cell Biology, Rice University, Houston, Texas, USA Current Biology, in press One mechanism multicellular structures use for controling cell number [1, 2] involves the secretion and sensing of a factor, like leptin [3] or myostatin [4] in mammals. Dictyostelium cells secrete autocrine factors for sensing cell density prior to aggregation and multicellular development [5, 6]. One of these factors, CMF (conditioned medium factor), is a glycoprotein that enables starving cells to respond to cAMP pulses [7, 8, 9]. Its actions are mediated by two receptors. CMFR1 activates a G-protein independent signaling pathway that regulates gene expression [10]. An unknown GÄ„1-dependent receptor activates phospholipase C (PLC), which, by an unknown mechanism, regulates the lifetime of GÄ„2-GTP [11, 12, 13]. Here we describe RpkA, an unusual seven transmembrane receptor fused to a C-terminal PIP5 kinase domain that localizes in membranes of a late endosomal compartment. Loss of RpkA resulted in formation of persistent loose aggregates concomitant with altered expression of cAMP-regulated genes. The developmental defect can be rescued by full length RpkA and the transmembrane domain only. The PIP5 kinase domain, although important for efficient targeting, is dispensable for the developmental role of RpkA. rpkAÄ{ cells secrete and bind CMF but are unable to induce downstream responses like production of inositol trisphosphate. Inactivation of Ga1, a negative regulator of CMF signaling, rescued the developmental defect of the rpkAÄ{ cells, suggesting that RpkA actions are mediated by this G protein. RpkA constitutes an essential component of the cell density sensing mechanism that regulates aggregation during Dictyostelium development. Submitted by: Angelika A. Noegel [noegel@uni-koeln.de] -------------------------------------------------------------------------------- Desalted deep sea water increases transformation and homologous recombination efficiencies in Dictyostelium discoideum Hidekazu Kuwayama and Akira Nagasaki Journal of Molecular Microbiology and Biotechnology, in press The life cycle of Dictyostelium discoideum consists of many cellular and developmental aspects. By virtue of its relatively high transformation efficiency and a small haploid genome, this organism has proven to be advantageous for characterizing gene functions. However, a much higher transformation efficiency is required as one of the prerequisites for unravelling gene function on a genome-wide scale. In this study, we describe the positive effect of desalted deep sea water, when used as a solvent medium, on the transformation and homologous recombination efficiencies in Dictyostelium. A standard Dictyostelium medium HL5 containing desalted deep sea water, HL5dsw, distinctly increased both the transformation and homologous recombination efficiencies by approximately 2- to 3-fold. Furthermore, we observed that the growth of cells in HL5dsw both before and after electroporation contributed to the increase in transformation efficiency. These results indicate that a simple modification of the solvent medium remarkably enhanced the isolation of transformants and gene targeted clones, which had previously been difficult to isolate. Submitted by: Hidekazu Kuwayama [hidekuwayama@biol.tsukuba.ac.jp] -------------------------------------------------------------------------------- Evolution of size and pattern in the social amoebas Review Pauline Schaap College of Life Sciences, University of Dundee, UK. Bioessays, in press A fundamental goal of biology is to understand how novel phenotypes evolved through changes in existing genes. The dictyostelids or social amoebas represent a simple form of multicellularity, where starving cells aggregate to build fruiting structures. This review summarizes efforts to provide a framework for investigating the genetic changes that generated novel morphologies in the dictyostelids. The foundation is a recently constructed molecular phylogeny of the dictyostelids, which was used to examine trends in the evolution of novel forms and in the divergence of genes that shape these forms. There is a major trend towards formation of large unbranched fruiting bodies, which is correlated with the use of cyclic AMP (cAMP) as a secreted signal to coordinate cell aggregation. The role of cAMP in aggregation arose through co-option of a pathway that originally acted to coordinate fruiting body formation. The genotypic changes that caused this innovation and the role of dynamic cAMP signaling in defining fruiting body size and pattern throughout Dictyostelid evolution are discussed. Submitted by: Pauline Schaap [p.schaap@dundee.ac.uk] -------------------------------------------------------------------------------- Protocols for growth and development of Dictyostelium discoideum Petra Fey1, Anthony S. Kowal2, Pascale Gaudet1, Karen E. Pilcher1, and Rex L. Chisholm1,2* 1dictyBase, Center for Genetic Medicine, Northwestern University 2 Center for Genetic Medicine, Northwestern University Nat Protoc., in press Dictyostelium discoideum, a unicellular organism capable of developing into a multicellular structure, is a powerful model system to study a variety of biological processes. Because it is inexpensive and relatively easy to grow, Dictyostelium is also frequently used in teaching laboratories. Here we describe conditions for successfully growing and developing Dictyostelium cells and methods for long-term storage of Dictyostelium amoebae and spores. Submitted by: Rex Chisholm [r-chisholm@northwestern.edu] -------------------------------------------------------------------------------- Transformation of Dictyostelium discoideum with plasmid DNA Pascale Gaudet, Karen E. Pilcher, Petra Fey, and Rex L. Chisholm* dictyBase, 676 N. St. Clair St, Suite 1260, Chicago, IL, 60611 *corresponding author Nat Protoc., in press DNA-mediated transformation is one of the most widely used techniques to study gene function. The eukaryote Dictyostelium discoideum is amenable to numerous genetic manipulations that require insertion of foreign DNA into cells. Here we describe two commonly used methods to transform Dictyostelium cells: calcium phosphate precipitation, resulting in high copy number transformants, and electroporation, an effective technique for producing single integration events into genomic DNA. Single integrations are required for gene disruption by homologous recombination. We also discuss how different selection markers affect vector copy number in transformants and explain why blasticidin has become the preferred selectable marker for making gene knock-outs. Both procedures can be accomplished in less than two hours of hands-on time; however, the calcium-phosphate precipitation method contains several incubations, including one of at least four hours, so the total time required for the transformation is approximately 8 hours. Submitted by: Rex Chisholm [r-chisholm@northwestern.edu] -------------------------------------------------------------------------------- A reliable general purpose method for extracting genomic DNA from Dictyostelium cells. Karen E. Pilcher1, Petra Fey1, Pascale Gaudet1, Anthony S. Kowal2, and Rex L. Chisholm1,2* *corresponding author 1dictyBase, Center for Genetic Medicine, Northwestern University 2 Center for Genetic Medicine, Northwestern University *corresponding author Nat Protoc., in press In this protocol, we present a standard method for extracting DNA from cells of the social amoeba Dictyostelium discoideum. While this procedure is similar to other phenol:chloroform-based purification methods, it is modified to account for the high level of carbohydrate and nucleases found in Dictyostelium cells. Genomic DNA can be isolated from wild type and genetically modified cells using the described protocol, allowing molecular genetic analyses to be performed. Following cell lysis, nucleic acid extraction, and precipitation, the isolated DNA is suitable for digestion by restriction enzymes, amplification by PCR, and Southern blotting. This procedure takes approximately 3 hours to complete. Submitted by: Rex Chisholm [r-chisholm@northwestern.edu] -------------------------------------------------------------------------------- A general purpose method for extracting RNA from Dictyostelium cells Karen E. Pilcher, Petra Fey, Pascale Gaudet, and Rex L. Chisholm* dictyBase, 676 N. St. Clair St, Suite 1260, Chicago, IL, 60611 *corresponding author Nat Protoc., in press Here we present a protocol for the extraction of RNA from Dictyostelium discoideum. Dictyostelium is a social amoeba which undergoes a basic developmental program, and therefore analysis of RNA levels over a time course is a commonly used technique. This procedure is similar to other guanidine thiocyanate-based methods; however, it has been adjusted because of the large quantities of carbohydrate and nucleases found in Dictyostelium cells. After cell lysis and phenol:chloroform extraction, the resulting high quality RNA isolated with the described protocol allows the molecular genetic analysis of wild type and genetically modified cells. The purified RNA can be used for analyses such as Northern blotting, RT-PCR, and microarrays. From start to finish, this procedure requires approximately two hours to complete. Submitted by: Rex Chisholm [r-chisholm@northwestern.edu] ============================================================ [End dictyNews, volume 28, number 9]