CSM News Electronic Edition Volume 8, number 12 May 10, 1997 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" ============== Announcement ============== Dicty Investigator Database The online Dictyostelium Email directory has been upgraded to a database that can be modified through the web page. This database can be queried in a number of ways, such as to find a particular person, people at a particular University, or investigators in a particular city. The value of this database is up to YOU. Everyone who was on the email directory has an entry in the database. Please go to the database on the Dicty WWW Page and complete your entry by filling in your address and other information. You can modify or delete your entry at any time. The whole process is forms based and easy. Give it a try! If your email reader supports active links you should be able to just click on the following link: http://apps.basic.northwestern.edu/dicty/dictyqbe.html Please note that making an entry in the database DOES NOT alter your entry on the listserv. Changes or subscriptions to the listserv must still be made as decribed on the Dicty Web site. Please email any questions, comments or problems to dicty@nwu.edu. =========== Abstracts =========== Dictyostelium IQGAP-Related Protein Specifically Involved in the Completion of Cytokinesis Hiroyuki Adachi,1* Yasuhiro Takahashi,2 Takeshi Hasebe,2 Mikako Shirouzu,3 Shigeyuki Yokoyama,3,4 and Kazuo Sutoh1 1Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Meguro-ku, Tokyo 153, 2Department of Applied Chemistry, Kogakuin University, Shinjuku-ku, Tokyo 163-91, 3Cellular Signaling Laboratory, the Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-01, and 4Department of Biophysics and Biochemistry, School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan *Corresponding Author J. Cell Biol. in press The gapA gene encoding a novel RasGTPase-activating protein (RasGAP)-related protein was found to be disrupted in a cytokinesis mutant of Dictyostelium that grows as giant and multinucleate cells in a dish culture. The predicted sequence of the GAPA protein showed considerable homology to those of Gap1/Sar1 from fission yeast and the C-terminal half of mammalian IQGAPs, the similarity extending beyond the RasGAP-related domain. In suspension culture, gapA- cells showed normal growth in terms of the increase in cell mass, but cytokinesis inefficiently occurred to produce spherical giant cells. Time-lapse recording of the dynamics of cell division in a dish culture revealed that in the case of gapA- cells, cytokinesis was very frequently reversed at the step in which the midbody connecting the daughter cells should be severed. Earlier steps of cytokinesis in the gapA- cells seemed to be normal, since myosin II was accumulated at the cleavage furrow. Upon starvation, gapA- cells developed and formed fruiting bodies with viable spores, like the wild type cells. These results indicate that the GAPA protein is specifically involved in the completion of cytokinesis. Recently, it was reported that IQGAPs are putative effectors for Rac and CDC42, members of the Rho family of GTPases, and participate in reorganization of the actin cytoskeleton. Thus, it is possible that Dictyostelium GAPA participates in the severing of the midbody by regulating the actin cytoskeleton through an interaction with a member of small GTPases. --------------------------------------------------------------------- A novel prespore-cell-inducing factor in Dictyostelium discoideum induces cell division of prespore cells Akiko A. Oohata1, Manabu Nakagawa2, Masao Tasaka3* and Shigeru Fujii2 1Biological Laboratory and 2Chemical Laboratory, Kansai Medical University, Hirakata, Osaka 573, Japan 3National Institute for Basic Biology, Myodaiji, Okazaki 444, Japan *Present address: Department of Botany, Division of Biological Science, Graduate School of Science, Kyoto University, Kyoto 606-1, Japan Development, in press In Dictyostelium discoideum strain V12M2, most amoebae differentiate into prespore cells in a salt solution containing cAMP at a very low cell density (~102 cells/sq cm) if an adequately diluted conditioned medium (CM) is provided (Oohata, Differentiation (1995) 59, 283-288; this study). This finding suggests the presence of factor(s) released into the medium which are involved in inducing prespore cell differentiation. In the present study, we report the presence of two types of factors which function synergistically in prespore cell induction; one is a heat-stable and dialysable factor(s) and the other is a heat-labile and non-dialysable factor termed psi factor (pre-spore inducing factor). We purified and characterized psi factor. Its relative molecular mass was determined to be 106 kDa by SDS-PAGE and 180 kDa by gel filtration HPLC, respectively. These results indicate that psi factor exists as a dimer under native conditions. In addition to inducing prespore cell differentiation, psi factor induced cell division of prespore cells in submerged culture. Our results suggest that psi factor plays important roles not only in prespore cell differentiation but also in the progress of the cell cycle in the prespore pathway in normal development. --------------------------------------------------------------------- Dictyostelium RasG is Required For Normal Motility and Cytokinesis, But Not Growth Richard I. Tuxworth, Janet L. Cheetham, Laura M. Machesky, George B. Spiegelmann, Gerald Weeks and Robert H. Insall. Accepted, J. Cell Biol. RasG is the most abundant Ras protein in growing Dictyostelium cells, and the closest relative of mammalian Ras proteins. We have generated null mutants in which expression of RasG is completely abolished. Unexpectedly, RasG- cells are able to grow at nearly wild-type rates. However, they exhibit defective cell movement and a wide range of defects in the control of the actin cytoskeleton, including a loss of cell polarity, a lack of normal lamellipodia, formation of unusual small, punctate F-actin structures and a large number of abnormally long filopodia. Despite their lack of polarity and abnormal cytoskeleton, mutant cells perform normal chemotaxis. However, rasG- cells are unable to perform normal cytokinesis, becoming multinucleate when grown in suspension culture. Taken together, these data suggest a principal role for RasG in coordination of cell movement and control of the cytoskeleton. --------------------------------------------------------------------- Partial Inverse PCR (PI-PCR) - A new technique for cloning flanking sequences. Ka Ming Pang and David A. Knecht Department of Molecular and Cell Biology, University of Connecticut Biotechniques, In Press Two commonly used methods to obtain the flanking sequences of a fragment from genomic DNA are inverse PCR (IPCR)(2,4) and ligation-linker PCR (3). Each method has its advantages and limitations. Ligation-linker PCR requires proper ligation between oligomer linker or plasmid to genomic DNA fragments. The ratio of linker DNA and genomic DNA has to be serial diluted to obtain maximum intermolecular ligation (3). IPCR is relatively simple since intra-molecular ligation can be achieved at low DNA concentration. However, both methods require Southern blot analysis to map the restriction sites flanking the known sequence. In addition, the flanking DNA needs to be remapped for each step of a walk. We report here the development of partial inverse PCR (PI-PCR), a modification to IPCR that overcomes some of these limitations. For PI-PCR, a Sau 3A 1 (or other 4 base cutter) partial digest of genomic DNA is religated and used as a template for IPCR (Fig. 1). Since Sau 3A 1 sites are so common in most genomes, this approach eliminates the necessity to have any prior knowledge of restriction enzyme sites surrounding the gene of interest. --------------------------------------------------------------------- Differential Developmental Expression of the repB and repD Xeroderma Pigmentosum Related DNA Helicase Genes from Dictyostelium discoideum Sung-Keun Lee*, Sung-Lim Yu*, Ma. Xenia Garcia, Hannah Alexander and Stephen Alexander Division of Biological Sciences, University of Missouri, Columbia, MO 65211. Nucleic Acids Res., in press DNA helicases are essential to many cellular processes including recombination, replication and transcription, and some helicases function in multiple processes. The helicases encoded by the Xeroderma pigmentosum (XP) B and D genes function in both nucleotide excision repair and transcription initiation. Mutations that affect the repair function of these proteins result in XP while mutations affecting transcription result in neurological and developmental abnormalities, although the underlying molecular and cellular basis for these phenotypes is not well understood. To better understand the developmental roles of these genes, we have now identified and characterized the repB and repD genes from the cellular slime mold Dictyostelium discoideum. Both genes encode DNA helicases of the SF2 superfamily of helicases. The repD gene contains no introns and the repB gene contains only one intron, which makes their genomic structures dramatically different from the corresponding genes in mammals and fish. However the predicted Dictyostelium proteins share high homology with the human XPB and XPD proteins. The single copy of the repB and D genes map to chromosomes 3 and 1, respectively. The expression of repB and D (and the previously isolated repE) genes during multicellular development was examined, and it was determined that each rep gene has a unique pattern of expression, consistent with the idea that they have specific roles in development. The pattern and extent of expression of these genes was not affected by the growth history of the cells, implying that the expression of these genes is tightly regulated by the developmental program. The expression of the rep genes is a very early step in development and may well represent a key event in the initiation of development in this organism. --------------------------------------------------------------------- [End CSM News, volume 8, number 12]