CSM News Electronic Edition Volume 8, number 6 March 1, 1997 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmb.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmb.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmb.nwu.edu/dicty.html" =========== Abstracts =========== Chromosome Walking In Dictyostelium: An Application of the Single Specific Primer PCR. Michael P. Koonce Division of Molecular Medicine, Wadsworth Center, Empire State Plaza, Box 509 Albany, NY 12201-0509. J. Microbio. Meth., in press We describe here an application of the polymerase chain reaction that sidesteps the necessity of knowing sequence information at both ends of a desired fragment of genomic DNA. The methodology is straightforward and works efficiently in organisms with relatively simple genomes. We have used it to walk more than 8 kb in six steps from a "starter" DNA sequence that was identified in a cDNA library screen. This technology complements existing procedures to walk in either direction along chromosomes, but because of the multiple levels of selection employed, it may prove to be more effective. Its primary utility will probably lie in extending sequence information in simple organisms that do not have pre-existing cDNA or genomic libraries, or for rapidly isolating genomic sequences that flank a cDNA clone. --------------------------------------------------------------------- Biochemical Characterization of the Phagocytic Giant Cell Receptor for Dictyostelium discoideum Amoebae: Identification by Cell Blotting Keith E. Lewis and James C. Jamieson, BBRC, 230:505-508. A critical aspect of sexual development in Dictyoselium is the selection of "self" as a targeted food source by the phagocytic zygote giant cell (ZGC). It has previously been demonstrated that phagocytic giant cells preferentially take up amoebae of the same species suggesting that the process is mediated by a specific receptor. By using tunicamycin, which inhibits N-linked glycosylation and mevastatin and mevinolin, which both inhibit HMG-CoA-reductase, we have been able to further characterize the glycoprotein(s) involved in the recognition process. We have utilized a "cell blotting" technique which lends itself well to identifying glycoproteins which are present on the ZGC at the phagocytic stage and interactive with the target amoebae. The cell blotting data, combined with pharmacological evidence, identifies these glycoproteins as the receptors for cannibalistic phagocytosis by the ZGC of D. discoideum. --------------------------------------------------------------------- An in vivo approach for the identification of acceptor sites for O-glycosyltransferases: motifs for the addition of O-GlcNAc in Dictyostelium discoideum Eva Jung, Andrew A. Gooley, Nicolle H. Packer, Martin B. Slade, Keith L. Williams, and Werner Dittrich^n MUCAB (Macquarie University Center for Analytical Biotechnology), School of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia ^Present address: Hoechst AG, Frankfurt, Germany Biochemistry, in press. To identify and analyze acceptor sequences for O-glycosylation, we have developed an in vivo system expressing short peptides as glutathione-S-transferase fusion proteins in the eukaryotic host Dictyostelium discoideum. Using this approach we show that a short peptide motif (PTVTPT), present in the D. discoideum cell surface glycoprotein PsA, is sufficient as a signal for O-glycosylation, even when fused to a heterologous protein. Monosaccharide analysis and solid-phase protein sequencing showed that the modification is a single N-acetylglucosamine attached to threonine residues. This was further confirmed by electrospray-mass spectrometry. The O-linked glycosylation of both this peptide and authentic PsA presents the modB-dependent carbohydrate specific epitope identified by the monoclonal antibody MUD50. Substitution of threonine by serine residues in this peptide also yields a glycosylated fusion protein which is modified with single N-acetylglucosamine residues, but not all of the serines are glycosylated. --------------------------------------------------------------------- A site-specific DNA endonuclease specified by one of two ORFs encoded by a group I intron in Dictyostelium discoideum mitochondrial DNA Shinji Ogawa1, Kayo Naito1, Kiyohiko Angata1, Takahiro Morio1, Hideko Urushihara1 and Yoshimasa Tanaka1,2,* 1Institute of Biological Sciences and 2Center for TARA#, University of Tsukuba, Tsukuba, Ibaraki 305, Japan Gene, in press. The second intron (DdOX1/2.2) of Dictyostelium discoideum cytochrome oxidase subunit 1/2 fused gene has two free-standing ORF genes (Dd ai2a and Dd ai2b) in a loop, which have similar amino acid sequences and are homologous to aI4 DNA endonuclease (I-Sce II) of Saccharomyces cerevisiae. To elucidate the functions of these ORFs, we cloned the ORFs into an expression vector and introduced the composite vectors into E. coli. The expression of Dd ai2a in E. coli caused growth inhibition and degradation of the E. coli genomic DNA. To determine whether Dd ai2a protein is a homing type DNA endonuclease, the ability to cleave the homing site of its intron in vivo was examined. Dd ai2a cleaved only one strand of intronless DNA sequence at the site which coincides with the I-Sce II cleavage recognition site. We suppose that Dd ai2a functions actually as a homing type DNA endonuclease in D. discoideum mitochondria by virtue of other factors. To obtain further information about the relationship between the existence of introns and the mating system, we carried out in vitro self-splicing assay and polymerase chain reaction analysis using 13 strains of the cellular slime mold. --------------------------------------------------------------------- Sorting of the Initial Cell Types in Dictyostelium is dependent on the tipA gene Justin T. Stege, Gad Shaulsky and William F. Loomis Center for Molecular Genetics, Department of Biology University of California San Diego, La Jolla California 92093 Devel. Biol. in press About 8 hours after the initiation of development in Dictyostelium discoideum, a few randomly scattered cells express prestalk specific genes and subsequently sort out to the top of the aggregate where they form a tip. The tip elongates and forms the anterior of the migrating slug before differentiating into a stalk which supports the ball of spores in a mature fruiting body. Using REMI mutagenesis we isolated a mutant strain, AK244, in which the initial aggregate subdivides to give a highly papillated surface. This mutant fails to form slugs and appears to have a defect in sorting of prestalk cells. The disrupted gene, tipA, encodes a novel 83kDa protein and is preferentially expressed in PST-O cells after the cell types have sorted out. Mutant strains that lack TipA express the prestalk specific gene ecmA at reduced levels and form very few spores. These defects cannot be overcome by developing the mutant cells in the presence of wild-type cells. Thus, TipA acts in a cell-autonomous manner at an early stage in development. Using strains carrying reporter constructs, we found that mutant cells expressing a prestalk marker remain dispersed in the aggregates. Prespore cells appear to sort such that the base is free of cells expressing cell type specific markers. Even after 20 hours of development, when wild type cells are undergoing terminal differentiation, prestalk cells in tipA- mutants form very small clumps, most of which fail to sort to the periphery or the tops of aggregates. The tipA gene appears to play an essential role in the sorting of the initial cell types. --------------------------------------------------------------------- [End CSM News, volume 8, number 6]