CSM News Electronic Edition Volume 5, number 4 August 5, 1995 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 at the URL "http://worms.cmsbio.nwu.edu/dicty.html" ============================== In Situ Hybridization Update ============================== Bill Loomis has provided the following update to the In Situ Hybridization Protocol. RIBOPROBES FOR WHOLE-MOUNT IN SITU LABELLING OF DICTYOSTELIUM Ricardo Escalante and William F. Loomis We have found that using RNA probes rather than DNA probes improves the signal-to-noise for whole-mount in situ hybridization of low abundance mRNAs expressed during development of Dictyostelium. Samples were fixed and prepared for hybridization exactly as described before (Escalante and Loomis, Develop. Biol. in press). However, the temperature was raised to 50degC during the prehybridization and hybridization steps as well as during the final wash step which done with 0.1 x SSC. Probe labeling Riboprobes were labelled with digoxigenin-UTP by in vitro transcription using the Dig RNA Labeling Kit from Boehringer (Cat. No. 1175025) following the manufacturer' s instructions. Labeled RNA was partially hydrolized as suggested by O' Neill, J.W. and Bier, E. (1994). BioTechniques 17(5) 870-875. [0.1M carbonate pH 10, 65degC, 20 min]. These modifications give more sensitivity and have been successfully used to detect low abundant transcripts such as those from the prestalk specific tagB gene. We have also found that fixed slugs can be stored in methanol at -20degC for at least two weeks and still give clear signals concerning cell type localization of specific mRNAs. ========== Abstract ========== TARGETED DISRUPTION OF THE DICTYOSTELIUM MYOSIN ESSENTIAL LIGHT CHAIN GENE PRODUCES CELLS DEFECTIVE IN CYTOKINESIS AND MORPHOGENESIS Tung-Ling L. Chen, Patricia A. Kowalczyk, Guyu Ho and Rex L. Chisholm* Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611 J. Cell Sci., in press. We have previously demonstrated that the myosin essential light chain (ELC) is required for myosin function in a Dictyostelium cell line, 7-11, in which the expression of ELC was inhibited by antisense RNA overexpression [Pollenz et al Cell 69, 951-962 1992]. We have now disrupted the gene encoding the ELC (mlcE) in Dictyostelium by gene targeting. The mlcE- mutants provide a clean genetic background for phenotypic analysis and biochemical characterization by removing complications arising from the residual ELC present in 7-11 cells, as well as the possibility of mutations due to insertion of the antisense construct at multiple sites in the genome. The mlcE- mutants, when grown in suspension, exhibited the typical multinucleate phenotype observed in both myosin heavy chain mutants and 7-11 cells. This phenotype was rescued by introducing a construct that expressed the wild type Dictyostelium ELC cDNA. Myosin purified from the mlcE- cells exhibited significant calcium ATPase activity, but the actin-activated ATPase activity was greatly reduced. The results obtained from the mlcE- mutants strengthen our previous conclusion based on the antisense cell line 7-11 that ELC is critical for myosin function. The proper localization of myosin in mlcE- cells suggests that its phenotypic defects primarily arise from defective contractile function of myosin rather than its mislocalization. The enzymatic defect of myosin in mlcE- cells also suggests a possible mechanism for the observed chemotatic defect of mlcE- cells. We have shown that while mlcE- cells were able to respond to chemoattractant with proper directionality, their rate of movement was reduced. During chemotaxis, proper directionality toward chemoattractant may depend primarily on proper localization of myosin, while efficient motility requires contractile function. In addition, we have analyzed the morphogenetic events during the development of mlcE- cells using lacZ reporter constructs expressed from cell type specific promoters. By analyzing the morphogenetic patterns of the two major cell types arising during Dictyostelium development, prespore and prestalk cells, we have shown that the localization of prespore cells is more susceptible to the loss of ELC than prestalk cells, although localization of both cell types is abnormal when developed in chimeras formed by mixing equal numbers of wild type and mutant cells. These results suggest that the morphogenetic events during Dictyostelium development have different requirements for myosin function. -------------------------------------------------------------------------- Sequence and developmental regulation of the gene that encodes the Dictyostelium discoideum L3 ribosomal protein Laura F. Steel, Polly D. Farnum, and Priya Kunapoli Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA Gene, in press SUMMARY We have isolated and characterized genomic and cDNA recombinant plasmids encoding the Dictyostelium discoideum ribosomal protein L3 (rpL3). Genomic plasmids were identified using a probe derived from the Saccharomyces cerevisiae TCM1 gene, that encodes the yeast rpL3. The D. discoideum rpL3 gene (DdL3) contains two introns and encodes a protein 398 amino acids in length that shows a high degree of homology to the conserved rpL3 protein of both lower and higher eukaryotes. During development, both the pattern of accumulation of DdL3 mRNA and changes in its translational activity are identical to those observed for other r-protein mRNAs. --------------------------------------------------------------------------- [End CSM News, volume 5, number 4]