Dicty News Electronic Edition Volume 10, number 5 February 14, 1998 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@nwu.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" =========== Abstracts =========== A ribosomal protein gene cluster is encoded in the mitochondrial DNA of Dictyostelium discoideum: UGA termination codons and similarity of gene order to Acanthamoeba castellanii Masao Iwamoto1, Min Pi, Mika Kurihara, Takahiro Morio, Yoshimasa Tanaka* Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan Curr. Genet. accepted Abstract We sequenced a region of about 14.5 kb downstream from the ribosomal protein L11 gene (rpl11) in the mitochondrial DNA (54 kb) of the cellular slime mold, Dictyostelium discoideum. Sequence analysis revealed that ten ribosomal protein genes and six open reading frames (ORFs) formed a cluster arranged in the order: rpl11-orf189-rps12-rps7-rpl2-rps19-orf425-orf1740-rpl16-rpl14- orf188-rps14-rps89-rps12-rps7-rpl2-rps19-orf425-orf1740-rpl16- rpl14-orf188-rps14-rps8-rpl6-rps137-rpl2-rps19-orf425-orf1740- rpl16-rpl14-orf188-rps14-rps8-rpl6-rps13-orf127-orf79-orf425- orf1740-rpl16-rpl14-orf188-rps14-rps8-rpl6-rps13-orf127-orf796. The order was very similar to that of homologous genes in Acanthamoeba castellanii mitochondrial DNA. The N-terminal region of the ORF425 and the C-terminal region of the ORF1740 had partial similarities to ribosomal protein S3 of other organisms. The termination codons of rpl16 and orf188 were UGA, which has not hitherto been found in genes encoded in D. discoideum mitochondrial DNA. ------------------------------------------------------------------------- Modulation of actin affinity and actomyosin ATPase by charge changes in the myosin motor domain. Max-Planck-Institut für Medizinische Forschung, Heidelberg and Max-Planck-Institut für Molekulare Physiologie, Dortmund (#) Marcus Furch, Michael A. Geeves (#), and Dietmar J. Manstein Biochemistry, in press ABSTRACT: The effects of mutations in an actin-binding surface loop of myosin (loop2) are described. Part of loop 2, the segment between myosin residues 618 and 622, was replaced with sequences enlarged by the introduction of positively charged GKK- or neutral GNN-motifs. Constructs with loops carrying up to 20 additional amino-acids and charge variations from -1 to +12 were produced. Steady state and transient kinetics were used to characterize the enzymatic behavior of the mutant motor domains. Binding of nucleotide was not affected by any of the alterations in loop 2. In regard to their interaction with actin, constructs with moderate charge changes (-1 to +2) displayed wild type-like behavior. Introduction of more than one GKK-motif led to stronger coupling between the actin- and nucleotide-binding sites of myosin and an up to 1,000-fold increased affinity for actin in the absence of ATP and at zero ionic strength. In comparison to the wild type construct M765, constructs with 4 to 12 extra charges displayed an increased dependence on ionic strength in their interaction with actin, a 2- to 3-fold increase in kcat, a more than 10-fold reduction in Kapp for actin, and a 34- to 70-fold increase in catalytic efficiency. ------------------------------------------------------------------------- Preferential expression of the cDNA encoding the proteasome subunit during the growth / differentiation transition of Dictyostelium cells Soo-Cheon Chae and Yasuo Maeda * Biological Institute, Graduate School of Science, Tohoku University, Aoba, Sendai 980-8578, Japan Biochem. Biophys. Res. Commun., in press Abstract: A proteasome subunit-1 gene (DAPS-1) was isolated as one preferentially expressed during the transition from growth to differentiation in Dictyostelium discoideum cells, using the differential display method. The DAPS-1 cDNA sequence with a length of 882 bp encodes a protein (Mr. 23.4 kDa) consisting of 213 amino acids. The deduced amino acid sequence of DAPS-1 showed 61% and 58% identity to the proteasome subunit Y of Xenopus laevis and Homo sapiens, respectively and 48% and 47% identity to the proteasome subunit LMP2 of Homo sapiens and Orizas latipes, respectively. Northern analysis revealed that a 1.0 kb of DAPS-1 mRNA is predominantly expressed during the early stage of differentiation induced by starvation. This seems to indicate that the DAPS-1 protein may be involved in proteolysis coupled with active exchange of the cellular protein composition during the phase-shift of Dictyostelium cells from the proliferative to differentiated state. ------------------------------------------------------------------------- [End Dicty News, volume 10, number 5]