Cellular Slime Mold Electronic Newsletter Volume 1, Issue #1 May 15, 1993 This is the first issue of the Electronic Version of the Cellular Slime Mold Newsletter. The purpose is to efficiently distribute information of interest to the community of folks who use cellular slime molds as an experimental organism. Abstracts or other items for inclusion in the newsletter should be submitted by e-mail to r-chisholm@nwu.edu. --------------------------------------------------------------------- The Highly Divergent alpha and beta -Tubulins From Dictyostelium discoideum are Encoded by Single Genes. Leda Trivinos-Lagos, Tetsuo Ohmachi , Caryn Albrightson, Roy G. Burns, Herbert L. Ennis, and Rex L. Chisholm Accepted. J. Cell Science. As a step in the characterization of the microtubule system of Dictyostelium discoideum, we have isolated and sequenced full-length cDNA clones which encode the Dictyostelium alpha- and beta- tubulins, as well as the Dictyostelium alpha-tubulin gene. Southern blot analysis suggests that Dictyostelium is unusual in that its genome contains single alpha- and beta-tubulin genes, rather than the multi-gene family common in most eukaryotic organisms. The complete apha-tubulin cDNA contains 1558 nucleotides with an open reading frame which encodes a protein of 457 amino acids. The complete beta-tubulin cDNA contains 1572 nucleotides and encodes a protein of 456 amino acids. Analysis of the deduced protein sequences indicates that while there is a significant degree of sequence similarity between the Dictyostelium tubulins and other known tubulins, the Dictyostelium alpha-tubulin displays the greatest sequence divergence yet described. Single alpha- and beta-tubulin transcripts are detected by Northern blot analysis during all stages of Dictyostelium development. The highest levels of message accumulate late in germinating spores and vegetative amoebae. Despite changes in alpha- and beta-tubulin mRNA levels, protein levels remain constant throughout development. We have expressed the carboxy terminal 2/3 ofthe alpha- and beta-tubulins as trpE fusions in Escherichia coli and used this protein to produce polyclonal antisera specific for the Dictyostelium alpha- and beta-tubulins. These antisera recognize one alpha- and two beta-tubulin spots on Western blots of 2-D gels and, by indirect immunofluorescence, both recognize the interphase and mitotic microtubule arrays in vegetative amoebae. --------------------------------------------------------------------- coactosin, A 17 kDa F-ACTIN BINDING PROTEIN FROM DICTYOSTELIUM DISCOIDEUM E. L. de Hostos*, B. Bradtke, F. Lottspeich, and G. Gerisch, Max-Planck-Institut fur Biochemie W-8033 Martinsried, Germany *Corresponding author, present address: Department of Pharmacology University of California San Francisco, CA, 94143-0450 Accepted, Cell Motility and the Cytoskeleton Absract A 17 kDa protein, designated as coactosin, has been purified from an actin-myosin complex reconstituted in vitro from a soluble fraction of Dictyostelium discoideum cells. The protein binds to F-actin in vitro without significantly altering its viscosity. Immunoblots labeled with monoclonal antibodies indicate that part of the protein is associated with the detergent-insoluble cytoskeleton. cDNA clones comprising the entire coding region of coactosin have been isolated from an expression library. The cDNA-derived amino-acid sequence reveals similarities of coactosin to the drebrins identified in neurons and to actin-binding proteins from other organisms, including yeast ABP1p, and yeast and vertebrate cofilins. --------------------------------------------------------------------- Vasu, S., Kedersha, N. and Rome, L.H. cDNA cloning and disruption of the major vault protein alpha gene in Dictyostelium discoideum J. Biol.Chem. (1993) In Press. Abstract Vaults are large cytoplasmic ribonucleoprotein particles found in nearly all eukaryotic cells. Dictyostelium vaults contain two major proteins, MVP alpha (94.2 kDa) and MVP beta (approx. 92 kDa). Using an anti-rat vault antibody we screened a Dictyostelium cDNA expression library and isolated a 2.8 kb clone which contained a single full-length reading frame. The identity of the clone was established by the presence of a predicted 20 amino acid sequence identical to that found in a peptide sequenced from purified MVP alpha. We have disrupted the single copy gene using homologous recombination and have demonstrated a loss of MVP alpha. Although the cells still produce MVP beta, they do not contain characteristic vault particles suggesting that MVP alpha is required for normal vault structure. These cells should be a valuable tool for elucidating the function of vaults. ----------------------------------------------------------------------- [END Volume 1, Issue #1 CSM Electronic Newsletter]