CSM News Electronic Edition Volume 3, number 16 November 5, 1994 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 [165.124.233.50], via Gopher at the same address, or by World Wide Web through www.nwu.edu. Wolfgan Nellen writes: hello out there! I am looking for a paper on DNA methylation in Dicty. As far as I remember there was a report a year or two ago stating that there was no detectable CpG methylation. Could anybody give me this reference without putting in too much of an effort? Is there anything more recent? Thanks a lot! best regards w message from: wolfgang nellen MPI Biochem. D-82152 Martinsried Germany Tel. ++49 (89) 8578 2241 FAX ++49 (89) 8578 3777 nellen@vms.biochem.mpg.de =========== Abstracts =========== Specific expression of a gene encoding a novel calcium-binding protein, CAF-1, during transition of Dictyostelium cells from growth to differentiation Fumiyoshi Abe and Yasuo Maeda Biological Institute, Faculty of Science, Tohoku University, Aoba, Sendai 980-77, Japan Development Growth & Differentiation, in press. SUMMARY The gene expressions involved in the transition from cell proliferation to differentiation were analyzed, using synchronized Dictyostelium discoideum Ax-2 cells and the differential plaque hybridization method. As one of genes (cDNAs) specifically expressed when Ax-2 cells were starved just before the PS-point (putative shift point; a switchover point from growth to differen- tiation in the cell cycle), calfumirin-1 (CAF-1) was cloned, which encoded a novel calcium-binding protein with E-F hand. Although CAF-1 mRNA was slightly expressed in vegetatively growing cells, the expression was markedly increased in response to starvation of cells just before the PS-point. Northern analysis using non-synchronized Ax-2 cells showed that the CAF-1 mRNA is predominantly expressed within a few hours of starvation. Such a starvation-induced early expression of the CAF-1 mRNA raised a possibility that CAF-1 might be one of calcium-binding proteins involved in the phase-shift of cells from growth to differentiation. --------------------------------------------------------------------- A proteinous factor mediating intercellular communication during the transition of Dictyostelium cells from growth to differentiation Norio Iijima, Takashi Takagi, and Yasuo Maeda Biological Institute, Faculty of Science, Tohoku University, Aoba, Sendai 980-77, Japan Zoological Science, in press. In general, cell differentiation and proliferation are mutually exclusive. Transition of the cellular slime mold Dictyostelium discoideum from growth to differentiation is triggered mainly by a secreted factor(s) in addition to nutritional deprivation. To purify and identify the factor required for the growth/differentiation transition, a new assay system was designed. Under low- nutrient conditions, cells could grow to multiply, but never developed. The cellular development including aggregation, however, was induced by the addition of conditioned medium (CM) in which growing or starving Dictyostelium cells had been cultured. The CM inhibited the synthesis of nuclear DNA and induced the cells to acquire chemotactic competence to cAMP, thus suggesting the presence of a secreted factor(s) required for growth/differentiation transition in the CM. The active factor(s) in CM (referred to as CMF450; conditioned medium factor) was found to be sensitive to heat and have a large molecular size. The CMF450 was purified and identified to be a proteinous macromolecule of Mr 450kDa, which was mainly composed of 94kDa, 79kDa, and 49kDa subunits under a native condition. -------------------------------------------------------------------- The pH-senstive actin-binding protein hisactophilin of Dictyostelium exists in two isoforms which both are myristylated and distributed between between plasma membrane and cytoplasm. F. Hanakam, C. Eckerskorn, F. Lottspeich, A. Mueller-Taubenberger, W. Schaefer, and G. Gerisch. J. Biol. Chem., in press. SUMMARY The histidine-rich protein hisactophilin is known to be associated with the inner surface of the plasma membrane and to be present as a soluble protein in the cytoplasm of Dictyostelium discoideum cells. Mass spectrometry of hisactophilin from the cytosol or extracted from a membrane fraction showed that none of the hisactophilin purified from D. discoideum cells had the mass predicted from the known cDNA-derived sequence of the protein. ES-MS and LSI-MS of tryptic fragments separated by reversed phase HPLC identified the most hydrophobic peptide as a myristylated fragment from the N-terminus of hisactophilin. Taken together the analytical data, it is concluded that all hisactophilin in D. discoideum cells is N-terminally modified by myristylation. By reversed-phase HPLC, two isoforms of hisactophilin, Hs I and Hs II, were recovered from the cytosolic as well as the membrane fraction of D. discoideum cells. Whereas the masses of Hs I fragments produced by trypsin fit into the previously published sequence of hisactophilin (myristylation considered), Hs II is another protein distinguished from Hs I by several amino-acid exchanges. Hs I and Hs II can form homo- and heterodimers by disulfide bridges. Hisactophilin is phosphorylated in vivo. Both isoforms proved to be sustrates of membrane-associated threonine/serine kinase from D. discoideum, which may regulate the interaction of hisactophilin with the plasma membrane. --------------------------------------------------------------------- [End CSM-News, vol. 3, number 16]