CSM News Electronic Edition Volume 1, number 17 October 2, 1993 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [129.105.233.50]. =================== Positions Available =================== FACULTY POSITIONS in CELL BIOLOGY Division of Biological Sciences, University of Missouri-Columbia The Division of Biological Sciences continues a multi-year hiring program with two tenure-track faculty positions to be filled at the assistant professor level with individuals using molecular methods to investigate basic problems in cell biology, including but not limited to protein trafficking and secretion, neurotransmission, cytoskeletal organization, cell motility, organelle biogenesis, signal transduction and cell cycle control. Individuals using yeast as an experimental organism and those studying problems of neurobiology (vertebrate or invertebrate) are of particular interest to complement existing programs, although other areas will be considered. The Division offers highly competitive salaries, generous start-up packages, modern research laboratories and support facilities, an active graduate program with institutional support for students and postdoctoral associates, and an interactive faculty. The Division is firmly committed to fostering ethnic and racial diversity on its faculty. Applications from women and members of minority groups are strongly encouraged. Applicants with a PhD, postdoctoral research experience, and a demonstrated potential for creative research should send their curriculum vitae, reprints of representative publications, statement of future research and teaching interests, and three letters of reference to: Dr. John David Division of Biological Sciences 110 Tucker Hall University of Missouri Columbia, Missouri 65211 Applications must be received by November 15, 1993 to receive full consideration. =========== Abstracts =========== Molecular Cloning and the C-terminal Processing of Gp64, a Putative Cell-Cell Adhesion Protein of the Cellular Slime Mold Polysphondylium pallidum Ri-ichiroh Manabe (1), Tamao Saito (2), Takashi Kumazaki (3), Takehisa Sakaitani (2), Nobuyuki Nakata (2), and Hiroshi Ochiai (2) (1):Research Institute, Osaka Medical Center for Maternal and Child Health, (2):Department of Biology, Faculty of Science, Hokkaido University, Sapporo, 060 Japan, (3):Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, 060 Japan Journal of Biological Chemistry, in press. The cellular slime mold Polysphondylium pallidum expresses a cell-surface glycoprotein (referred to as gp64), which seems to be implicated in cell-cell adhesion. We identified a near full-length gp64 cDNA (1,104 base) upon screening a P. pallidum lambda gt11 library with a monoclonal antibody. The open reading frame encodes a protein of 320 amino acids with a molecular mass of 32,752 Da; the protein includes hydrophobic segments at both a NH2- and a COOH-terminal ends. By an Edman degradation analysis of S-pyridylethylated gp64 and its COOH-terminal peptide, it was found that the NH2-and COOH-terminal segments are both removed from the precursor protein of gp64. The COOH-terminal segment was isolated from a lysylendopeptidase digest of gp64 by a affinity method. The COOH-terminal segment was identified at positions 266-279 in the primary sequence deduced from the cDNA sequence. The mature gp64 consisted of 279 amino acid residues, and extremely rich in Cys residues (36 Cys / 279 amino acids=12.9%). Although there was already maximally accumulation of gp64 mRNA in vegetative cells, the protein reached a maximal level during aggregation stage, decreased and then leveled off through the developmental cycle. ---------------------------------------------------------------------- Evidence for a glycolipid anchor of gp64, a putative cell-cell adhesion protein of Polysphondylium pallidum Tamao SAITO and Hiroshi OCHIAI Department of Biology, Faculty of Science, Hokkaido University, Sapporo, 060 Japan European Journal of Biochemistry, in press. The membrane-bound glycoprotein (gp64) of the cellular slime mold, Polysphondylium pallidum, is a putative cell-cell adhesion protein identified by adhesion-blocking antibody fragments (Fab). Since gp64 can be purified in a few days and in substantial yields, it is a good candidate for clarifying the structure of a cell-cell adhesion protein. This paper reveals that gp64 possesses a glycolipid anchor which is sensitive to deamination but resistant to phosphatidylinositol specific phospholipase C (PtdIns-PLC; EC 3. 1. 4. 10) of Bacillus thuringiensis . Although the anchor resistance to PtdIns-PLC could be ascribed to the presence of an additional acyl chain on the inositol ring in the glycosyl phosphatidylinositol ((Ose) n-PtdIns)-anchors, this was not the case. After a mild-base treatment that released an additional acyl chain on the inositol ring, only a trace amount of fatty acid was detected, but , after strong acid hydrolysis, we detected both amide-linked fatty acids and a long chain base. The long chain base was further analysed by gass-chromatography / mass spectrometry (GC-MS) and was found to be phytosphingosine. Both fatty acids and myo -inositol were also analysed by gass-chromatography / mass spectrometry. These data suggest that gp64 possesses a glycolipid anchor which contains ceramide and myo -inositol. ---------------------------------------------------------------------- Functional Analysis of a Cardiac Myosin Rod in Dictyostelium discoideum. Janine M. LeBlanc-Straceski*, Yoshio Fukui, Regina L. Sohn, James A. Spudich, Leslie A. Leinwand. Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Cell Motility and the Cytoskeleton, in press. Manipulation of the single conventional myosin heavy chain (mhc) gene in Dictyostelium discoideum (Dd) has delineated an essential role for the filament-forming, or light meromyosin (LMM) domain of the myosin molecule in cytokinesis, development, and in the capping of cell surface receptors (see Spudich, 1989; Egelhoff et al 1991). In order to assess the functional relationship between sarcomeric and cytoplasmic myosins, a chimeric gene encoding the Dd myosin head and subfragment 2 fused to rat b cardiac LMM was transfected into both wild type and mhc null Dd cells. Chimeric myosin was organized into dense cortical patches in the cytoplasm of both wild type and Dd mhc null cells. Although null cells expressing chimeric mhc at ~10% of Dd mhc levels were unable to grow in shaking suspension or to complete development, chimeric myosin was able to rescue capping of cell surface receptors, to associate with filamentous actin, and to localize to the correct subcellular position during aggregation. Deletion of 29 amino acids in the rod corresponding to a previously defined filament assembly competent region eliminated the cortical patches and the posterior localization during chemotaxis. Taken together, these observations suggest that sarcomeric and cytoplasmic myosin rods are functionally interchangeable in several aspects of nonmuscle motility. ------ [[End CSM-News, volume 1, number 17]]