CSM News Electronic Edition Volume 7, number 1 July 6, 1996 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmb.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmb.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmb.nwu.edu/dicty.html" =========== Abstracts =========== Temporal Regulation of the DICTYOSTELIUM Glycogen Phosphorylase 2 Gene Charles L. Rutherford*, Ornella Selmin, and Sandra Peters-Weigel Biology Department, Molecular and Cellular Biology Section, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, Biochem. Biophys. Acta, in press. The product of the glycogen phosphorylase-2 gene in Dictyostelium functions to provide the glucose units that are used to construct the structural components of the terminal stage of development. The nucleotide sequence of a genomic clone that extended from an Eco R1 site at -1,216 nucleotides from the translational start AUG codon (-930 nucleotides from the transcriptional start site) to a Rsa I site at +17 nucleotides is shown. We linked a 1,233 bp upstream gp2 fragment to a luciferase reporter gene in order to study the sequences that are involved in the temporal expression of the gene. Various deletions of the promoter-luciferase fusion were then transformed into Dictyostelium cells. All deletion constructs, from -1,216 to -486 nucleotides from the translational start codon, showed the same temporal pattern of expression as the authentic gp2 gene, as well as similar luciferase activities. Removal of an additional 37 nucleotides resulted in nearly 100 fold decrease in activity, yet retained the normal temporal expression of luciferase. In order to test if the fragment containing the "C and TAG" boxes was dependent on its position within the promoter, we amplified this segment and ligated it to an inactive construct. This fragment, extending from -461 to -282 bp and containing both TAG and C boxes, was fused to an inactive construct . This placed the TAG and C boxes 110 bp closer to the start site of translation than they were in the unmodified promoter, as well as deleted the segment from -282 to -172. This resulted in activation of the construct to a luciferase activity that corresponded to the active promoter, and enabled it to show temporal expression identical to the full length promoter. Analysis of DNA binding proteins with the gel shift assay revealed a stage dependent pattern of proteins that bound to the gp2 promoter. A similar pattern of temporal expression of the binding proteins was observed with either the full-length probe or with oligonucleotide probes that contained sequences that were identified as putative regulatory sites. Likewise, the full length and oligonucleotide probes demonstrated identical binding patterns during several steps of purification of the DNA binding proteins. SDS-PAGE and southwestern blot analysis of a DNA-affinity purified fraction, identified a 23kD peptide as the binding protein. --------------------------------------------------------------------- F. Rivero1, R. Furukawa2, A. A. Noegel1, and M. Fechheimer2# 1. Max-Planck-Institute for Biochemistry, 82152 Martinsried, FRG 2. Department of Cellular Biology, University of Georgia, Athens, Georgia Dictyostelium discoideum Cells Lacking the 34,000 Dalton Actin Binding Protein Can Grow, Locomote, and Develop, but Exhibit Defects in Regulation of Cell Structure and Movement: A Case of Partial Redundancy J. Cell Biol., in press. Abstract Cells lacking the Dictyostelium 34,000 dalton actin bundling protein, a calcium regulated actin cross-linking protein, were created in order to probe the function of this polypeptide in living cells. Cells lacking the 34 kD protein were obtained in strains derived from AX2 and AX3. Growth, pinocytosis, morphogenesis, and expression of developmentally regulated genes is normal in cells lacking the 34 kD protein. In chemotaxis studies, 34 kD- cells were able to locomote and orient normally, but showed an increased persistence of motility. The 34 kD- cells also lost bits of cytoplasm during locomotion. The 34 kD- cells exhibited either an excessive number of long and branched filopodia, or a decrease in filopodial length and an increase in the total number of filopodia per cell depending on the strain. Re-expression of the 34 kD protein in the AX2 derived strain led to a "rescue" of the defect in persistence of motility, and of the excess numbers of long branched filopodia, demonstrating that these defects are due to the absence of the 34 kD protein. We explain the results through a model of partial functional redundancy. Numerous other actin cross-linking proteins in Dictyostelium may be able to substitute for some functions of the 34 kD protein in the 34 kD- cells. The observed phenotype is presumed due to functions that cannot be adequately supplanted by a substitution of another actin cross-linking protein. We conclude that the 34 kD actin bundling protein is not essential for growth, but plays an important role in dynamic control of cell shape and cytoplasmic structure. --------------------------------------------------------------------- [End CSM-News, volume 7, number 7]