Dicty News Electronic Edition Volume 21, number 13 October 17, 2003 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 Dicty-News, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ============= Abstracts ============= Identification of Dictyostelium discoideum developmentally regulated genes whose expression is dependent on the MADS-box transcription factor SrfA Ricardo Escalante, Nicolas Moreno, and Leandro Sastre Eukaryotic Cell, in press The MADS-box transcription factor SrfA is required for spore differentiation in Dictyostelium discoideum. srfA null strains form rounded spores that do not resist adverse environmental conditions. Five genes whose expression is dependent on SrfA have been isolated by differential hybridization. One of these genes, sigC, is identical to phg1b, previously characterized in mutants with altered adhesive properties and found to encode a nine transmembrane domain protein. This gene is transcribed into two mRNAs, as the result of alternative splicing of two internal exons. The slower migrating mRNA codes for a shorter protein that lacks the first transmembrane fragment and is not expressed in srfA null strains. The other four genes (sigA, B, D and 45D) are expressed only during late developmental stages. In situ hybridization experiments showed that expression of sigA, B and D is restricted to the sorus of developing structures. sigA codes for a homolog of malate dehydrogenase that converts pyruvate to malate to replenish the tricarboxylic acid cycle. sigB encodes a protein with significant similarity to the GP63 metalloproteinase of Leishmania, leishmanolysin. The sequence of sigD is highly similar to that of several spore coat proteins of D. discoideum and it may play a role in that structure. The gene 45D codes for a RNA-binding protein homologue whose expression is also dependent on the GATA transcription factor stalky (StkA). The expression of sigB is also dependent on both SrfA and StkA. The expression of 45D, but not of sigA, B, C and D, can be induced in srfA null cells by constitutive PKA activation. Strains in which either sigA, sigB or sigD are disrupted were isolated and found to form spores that are not detectably different from those of wild type strains. Submitted by: Leandro Sastre [lsastre@iib.uam.es] ----------------------------------------------------------------------------- Regulatory Mechanism of Dictyostelium Myosin Light Chain Kinase A Hiroshi Tokumitsu*à, Naoya Hatano¤, Hiroyuki Inuzuka*, Yumi Ishikawa*, Taro Q. P. Uyeda¦, Janet L. Smith , and Ryoji Kobayashi* *Department of Signal Transduction Sciences and ¤Department of Cell Physiology, Kagawa Medical University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan, ¦Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8562, Japan,  Boston Biomedical Research Institute, 64 Grove St. Watertown, MA 02472-2829, U.S.A J. Biol. Chem., in press In this report, we have examined the activation mechanism of Dictyostelium MLCK-A by using constitutively active Ca2+/calmodulin-dependent protein kinase kinase (CaM-KKc) as a surrogate MLCK-A kinase. MLCK-A was phosphorylated at Thr166 by CaM-KKc, resulting in a ~140-fold increase in catalytic activity, using intact Dictyostelium myosin II. Recombinant Dictyostelium MRLC and Kemptamide were also readily phosphorylated by the activated MLCK-A. Mass spectrometry analysis revealed that E. coli-expressed MLCK-A was autophosphorylated at Thr289 and subsequent to Thr166 phosphorylation, MLCK-A also undergoes a slow rate of autophosphorylation at multiple Ser residues. Using site-directed mutagenesis, we showed that autophosphorylation at Thr289 is required for efficient phosphorylation and activation by an upstream kinase. By performing enzyme kinetics on a series of truncated MLCK-As, we found that residues 283-288 function as an autoinhibitory domain, and that autoinhibition is fully relieved by Thr166 phosphorylation. Simple removal of this region results in a significant increase in the kcat of MLCK-A, however, it does not generate maximal enzymatic activity. Together with the results of our kinetic analysis of the enzymes, these findings demonstrate that Thr166 phosphorylation of MLCK-A by an upstream kinase subsequent to autophosphorylation at Thr289 results in generation of maximum MLCK-A activity through both release of an autoinhibitory domain from its catalytic core and a further increase (15-19-fold) in the kcat of the enzyme. Submitted by: Janet Smith [smith@bbri.org] ----------------------------------------------------------------------------- Evolutionary questions raised by cellular slime mould development Sonia Kaushik(*) and Vidyanand Nanjundiah Indian Institute of Science, Bangalore 560012, India (*) Current address: Department of Microbiology, La Trobe University, VIC 3086, AUSTRALIA. Proceedings of the Indian National Science Academy, in press REVIEW The cellular slime moulds (CSMs) are amoeboid organisms whose life cycle can be viewed in two ways. Firstly, because free-living amoebae come together to build bodies, they are ideal models for studying multicellular development in terms of the properties of single cells. Secondly, coming together and participating in an integrated unit implies social behaviour. Consequently differentiation (especially in the advanced CSMs) can be seen as a form of division of labour in which only some amoebae get to transmit their genes to the next generation. Viewed thus, their life cycle is ideally suited for studying the evolutionary basis of cooperation with some members of the cooperating group exhibiting altruistic behaviour. The present review takes the second approach. We examine alternative explanations for social behaviour (i.e., multicellular development) based on individual-level and group (including kin-group) selection. Non-clonal fruiting bodies are likely to be common in nature; we show a case with at least nine genotypes. The CSMs display both individual and group-level adaptations and both levels of selection operate in their appropriate contexts. The review ends with questions for the future and indicates how studies of CSM development might help to explain the evolution of altruism in this group. Submitted by: Vidyanand Nanjundiah [vidya@ces.iisc.ernet.in] ----------------------------------------------------------------------------- Calcium regulates the expression of a Dictyostelium discoideum asparaginyl tRNA synthetase gene. Jyoti K. Jaiswal* and Vidyanand Nanjundiah Indian Institute of Science, Bangalore 560012, India. * (Current address) The Rockefeller University, 1230 York Avenue, Box 304, New York, NY 10021, USA Journal of Biosciences, in press In a screen for calcium-regulated gene expression during growth and development of Dictyostelium discoideum we have identified an asparaginyl tRNA synthetase (ddAsnRS) gene, the second tRNA synthetase gene identified in this organism. The gene shows many unique features. It is repressed by lowering cellular calcium, making it the first known calcium-regulated tRNA synthetase. Also, despite the calcium-dependence, its expression is unaltered during the cell cycle, making it the first D. discoideum gene to show a calcium-dependent but cell cycle phase-independent expression. Finally, the N-terminal domain of the predicted ddAsnRS protein shows higher sequence similarity to Gln tRNA synthetases than to other Asn tRNA synthetases. These unique features of the AsnRS from this primitive eukaryote point to a novel mechanism that regulates the components of the translation machinery and gene expression by calcium, and also indicate a link between the evolution of GlnRS and AsnRS in eukaryotes. Submitted by: Vidyanand Nanjundiah [vidya@ces.iisc.ernet.in] ----------------------------------------------------------------------------- A bZIP/bRLZ Transcription Factor Required for DIF Signaling in Dictyostelium Christopher R.L. Thompson (1), Qing Fu (1), Caroline Buhay (1), Robert R. Kay (2) and Gad Shaulsky(1) 1 Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, USA 77030 2 MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Development, in press The intermingled differentiation and sorting out of Dictyostelium prestalk-O and prespore cells requires the diffusible signaling molecule DIF-1 and provides an example of a spatial information-independent patterning mechanism. To further understand this patterning process, we used a genetic selection to isolate mutants in the DIF-1 response pathway. The disrupted gene in one such mutant, dimA-, encodes a bZIP/bRLZ transcription factor, which is required for every DIF-1 response investigated. Furthermore, the dimA- mutant shows strikingly similar developmental defects to the dmtA- mutant, which is specifically defective in DIF-1 synthesis. However, key differences exist: (1) the dmtA- mutant responds to DIF-1 but does not produce DIF-1 (2) the dimA- mutant produces DIF-1 but does not respond to DIF-1 (3) the dimA- mutant exhibits cell autonomous defects in cell type differentiation. These results suggest that dimA encodes the key transcriptional regulator required to integrate DIF-1 signaling and subsequent patterning in Dictyostelium. Submitted by: Gadi Shaulsky [gadi@bcm.tmc.edu] ----------------------------------------------------------------------------- Two Phases of Actin Polymerization Display Different Dependences on PI(3,4,5)P3 Accumulation and Have Unique Roles during Chemotaxis Lingfeng Chen, Chris Janetopoulos, Yi Elaine Huang, Miho Iijima, Jane Borleis and Peter N. Devreotes Department of Cell Biology, Johns Hopkins University, School of Medicine Baltimore, MD, 21205 MBC, in press The directional movement of cells in chemoattractant gradients requires sophisticated control of the actin cytoskeleton. Uniform exposure of D. discoideum amoebae as well as mammalian leukocytes to chemoattractant triggers two phases of actin polymerization. In the initial rapid phase, motility stops and the cell rounds up. During the second slow phase, pseudopodia are extended from local regions of the cell perimeter. These responses are highly correlated with temporal and spatial accumulations of PI(3,4,5)P3/PI(3,4)P2 reflected by the translocation of specific PH domains to the membrane. The slower phase of PI accumulation and actin polymerization is more prominent in less differentiated, unpolarized cells, is selectively increased by disruption of PTEN, and is relatively more sensitive to perturbations of PI3K. Optimal levels of the second responses allow the cell to respond rapidly to switches in gradient direction by extending lateral pseudopods. Consequently, PI3K inhibitors impair chemotaxis in wild type cells but partially restore polarity and chemotactic response in pten- cells. Surprisingly, the fast phase of PI(3,4,5)P3 accumulation and actin polymerization, which is relatively resistant to PI3K inhibition, can support inefficient but reasonably accurate chemotaxis. Submitted by: Lingfeng Chen [lche1@jhmi.edu] ----------------------------------------------------------------------------- Hypertonic signal promotes stability of Dictyostelium spores via a PKA independent pathway Yohko Yamada and Masazumi Sameshima The Tokyo Metropolitan Institute of Medical Science, Electron Microscopy Center FEMS Microbiol. Lett., in press Differentiation of Dictyostelium spores is initiated with rapid encapsulation of prespore cells under the control of cAMP dependent protein kinase (PKA), followed by further maturation process that involves cytoskeletal organization. Constitutive activation of PKA induces precocious formation of viable spores in development and confer cells the ability to encapsulate under specific submerged conditions. In this study we show that the stability of these spores depended on high osmotic condition during spore differentiation, indicating that hypertonic signal is required in addition to PKA to induce matured stable spores. Formation of stable spores under a high osmotic condition required high cell density, suggesting the involvement of additional cellular signaling. Submitted by: Yohko Yamada [yyamada@rinshoken.or.jp] ----------------------------------------------------------------------------- A/T-rich inverted DNA repeats are destabilized by chaotrope-containing buffer during purification using silica gel membrane technology. M. Prevorovsky and F. Puta BioTechniques 35:698-702 (October 2003) Ê The recovery of electrophoretically separated DNA fragment(s) from agarose gel is afrequently used method in most gene engineering procedures. These techniques wereconsiderably simplified by the implementation of commercial kits based on silicamicroparticles or glass fibers. In this report we show that during extraction the chaotropecontainingagarose-melting buffer can cause destabilization of short A/T rich DNA fragmentsof inverted repeat-structure. We propose that destabilized inverted repeats formed hairpinsafter the alleviation of denaturing conditions. Expression of RNA molecules of invertedrepeat-structure is the method of permanent gene silencing by RNA interference (RNAi) invarious organisms, so we believe that our findings are of interest to researchers conductingRNAi experiments and/or working with, e.g., Plasmodium and other unicellular pathogens, characterized by A/T rich genomes. Submitted by: Frantisek Puta [puta@natur.cuni.cz] =============================================================================== [End Dicty News, volume 21, number 13]