CSM News Electronic Edition Volume 1, number 19 October 23, 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]. --------------------------------------------------------------------- ABSTRACTS --------- Altered Morphology of Vegetative Amoebae Induced by Increased Expression of the Dictyostelium discoideum ras-Related Gene rap1. P.J. Rebstein, G.B. Spiegelman, and G. Weeks, Developmental Genetics, 1993. 14: p.347-355. Summary The rap1 gene of Dictyostelium discoideum is a member of the ras-gene superfamily of low molecular weight GTPase proteins. The rap1 gene is expressed both during growth and development in D. discoideum. To examine the action of the Rap1 protein in D. discoideum, the rap1 cDNA was expressed under the control of the inducible discoidin promoter. Treatment with conditioned media, which induces the discoidin promoter, increased Rap1 protein levels in vegetative cells approximately six fold. Overexpression of the Rap1 protein correlated with the appearance of morphologically aberrant vegetative amoebae: cells were extensively spread and flattened. The distribution of F-actin was altered in these cells, with an increase in actin staining around the cell periphery. Induction of the discoidin promoter by starvation in the rap1 transformants also resulted in spread flat cells. When starved D. discoideum amoebae are refed with HL5 media, the cells rapidly respond by rounding up. By contrast, the rap1 transformant cells showed a pronounced delay in rounding up. Rapid tyrosine phosphorylation of a p45 protein occurred in both control cells and the rap1 transformant upon refeeding, implying that the signal transduction pathway leading to tyrosine phosphorylation remained functional in the rap1 transformant. We propose that the Rap1 protein functions in the regulation of cell morphology in D. discoideum. ----------------------------------------------------------------------- Isolation of two novel ras genes in Dictyostelium discoideum; evidence for a complex, developmentally regulated ras gene subfamily. Juliet Daniel1, John Bush3, James Cardelli3, George B. Spiegelman1,2* & Gerald Weeks1,2 Departments of Microbiology1 and Medical Genetics2, University of British Columbia,Vancouver, B.C., V6T 1Z3, Canada and Department of Microbiology and Immunology3, Louisiana State University Medical Center, Shreveport, Louisiana, U.S.A. Oncogene, In Press Abstract: In Dictyostelium discoideum, three ras genes (rasD, rasG and rasB) and one ras-related gene (rap1) have been previously isolated and characterized, and the deduced amino acid sequence of their predicted protein products share at least 50% sequence identity with the human H-Ras protein. We have now cloned and characterized two additional members of the ras gene subfamily in Dictyostelium, rasC and rasS. These genes are developmentally regulated and unlike the previously isolated Dictyostelium ras genes, maximum levels of their transcripts were detected during aggregation, suggesting that the encoded proteins have distinct functions during aggregation. The rasC cDNA encodes a 189 amino acid protein that is 65% identical to the Dictyostelium RasD and RasG proteins and 56% identical to the human H-Ras protein. The predicted 194 amino acid gene product encoded by rasS is 60% identical to the Dictyostelium RasD and RasG proteins and 54% identical to the human H-Ras protein. Whereas RasD, RasG, RasB and Rap1 are totally conserved in their putative effector domains relative to H-Ras, RasC and RasS have single amino acid substitutions in their effector domains, consistent with the idea that they have unique functions. In RasC, aspartic acid-38 has been replaced by asparagine (D38N), and in RasS, isoleucine-36 has been replaced by leucine (I36L). In addition, both proteins have several differences in the effector-proximal domain, a domain which is believed to play a role in Ras target activation. In RasC, there is a single conservative amino acid change in the canonical sequence of the binding site for the Ras-specific monoclonal antibody Y13-259, and consequently, RasC is less immunoreactive with the antibody than either of the Dictyostelium RasD or RasG proteins. In contrast, RasS, which has three substitutions in the Y13-259 binding site, does not react with the Y13-259 antibody. ---------------------------------------------------------------------- Stereo-Electron Microscopy of DNA-Stained Mitotic Chromosomes from Dictyostelium Discoideum A. L. Olins and D. E. Olins The University of Tennessee - Oak Ridge Graduate School of Biomedical Sciences Box 2009, Biology Division, Oak Ridge National Laboratory Oak Ridge, TN 37831-8077, USA Cell Biology International, in press ABSTRACT: Mitotic chromosomes stained with uranyl and lead are difficult to visualize by electron micrsocopy (EM), but can be clearly seen with the DNA-specific reagent osmium ammine. In this case, mitotic chromosomes were stained with a modified reagent osmium ammine-B and examined by stereo-EM. DNA distribution in mitotic cells revealed unanticipated ultrastructural features: 1) mitotic chromosomes with surface loops of chromatin; 2) patches of condensed chromatin associated with the nuclear envelope. -------------------------------------------------------------------- Technical Tips -------------- Rich Sucgang writes: Anyone who has tried to PCR directly from Dicty cells knows that the raw cells won't work. Doing a miniprep is impractical for full scale screening if all you are doing is PCR. There are reports of using proteinase K or other proteinases to produce crude extracts that PCR. Actually, for the last 50 PCR runs, all that I did was wash a small number of cells, resuspend in 50 ul Sorensens buffer, and boil it for 5 min in a water bath at full boil. I then spin down the crud for 1-3 min, and take 5ul of the supernatant for PCR. Works so far. --------------------------------------------------------------------- [[End CSM-News, volume 1, number 19]]