CSM News Electronic Edition Volume 2, number 7 February 12, 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 [129.105.233.50], via Gopher at the same address, or by World Wide Web through WWW.acns.nwu.edu. ========== Abstracts ========== Analysis of a complex plasmid insertion in a phototaxis-deficient transformant of Dictyostelium discoideum selected on a Micrococcus luteus lawn. Z. Wilczynska and P.R. Fisher Department of Microbiology, La Trobe University, Bundoora 3083, Melbourne, Australia. Plasmid, in press. Summary A novel method for clonal selection of G418 resistant Dictyostelium discoideum transformants on lawns of Micrococcus luteus was developed. The procedure was used to isolate transformants deficient in phototaxis after non-targeted insertion of shuttle vector DNA. Southern blot analysis as well as restriction, T-tracking and sequencing analysis of plasmids rescued from the genomic DNA of one of the putative phototaxis gene disruptants showed that it contained a complex multicopy insertion of the vector. While insertions of such plasmid vectors might typically be in tandem multicopy format, they can be much more complex containing, as in this case, inverse as well as tandemly duplicated copies and various deletions. Note: For those people who requested a reference for citation purposes regarding the Micrococcus selection, this is it. The M. luteus strain is now freely available to all and can be obtained from Paul Fisher or from a number of other laboratories. --------------------------------------------------------------------- Production and Secretion of Recombinant Proteins in Dictyostelium discoideum Werner Dittrich, Keith L. Williams and Martin B. Slade School of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia Bio/Technology, in press SUMMARY We have expressed useful amounts of three recombinant proteins in a new eukaryotic host/vector system. The cellular slime mould Dictyostelium discoideum efficiently secreted two recombinant products, a soluble form of the normally cell surface asociated D. discoideum glycoprotein (PsA) and the heterologous protein glutathione-S-transferase (GST) from Schistosoma japonicum, while the enzyme (Beta)-glucuronidase (GUS) from Escherichia coli was cell associated. Up to 20mg/l of recombinant PsA and 1mg/l of GST were obtained after purification from a standard, peptone based growth medium. The secretion signal peptide was correctly cleaved from the recombinant GST- and PsA-proteins and the expression of recombinant PsA was shown to be stable for at least one hundred generations in the absence of selection. ----------------------------------------------------------------- The role of cGMP in photosensory and thermosensory transduction in Dictyostelium discoideum. P.K. DARCY, Z. WILCZYNSKA and P. R. FISHER Microbiology Department, La Trobe University, Bundoora 3083, Melbourne, Australia. Microbiology (formerly J. Gen. Micro), in press. SUMMARY Weak and strong light/heat stimuli induced changes in cyclic guanosine monophosphate (cGMP) levels in vegetative and aggregation competent amoebae and in slug cells of the Dictyostelium discoideum strain X22. Mutant strains derived from X22 with mutations in the phototaxis loci phoA-phoK fell into four phenotypic classes with respect to cGMP responses to weak and strong light/heat stimuli. These results suggest an intermediary role for cGMP in photosensory and thermosensory processing in slugs and amoebae. The streamer F mutant NP368 which has previously been shown to exhibit a prolonged cGMP response to cAMP, showed a wildtype cGMP response to light and heat. All phototaxis mutant strains with altered cGMP responses to light and heat were unaltered in their cGMP response to cAMP. These results suggest that cAMP and light/heat regulate cGMP via independent pathways. The thermotaxis mutant HPF228 showed altered cGMP responses to heat but not to light stimuli. This suggests that the mutation in HPF228 affects thermosensory transduction before convergence with the phototaxis pathway and the subsequent cGMP response. ------------------------------------------------------------------ Genetic analysis of Dictyostelium slug phototaxis mutants P.K. DARCY, Z. WILCZYNSKA and P. R. FISHER Microbiology Department, La Trobe University, Bundoora 3083, Melbourne, Australia. Genetics, in press. ABSTRACT Mapping and complementation analysis with seventeen phototaxis mutations has established eleven complementation groups phoA-phoK distributed over six linkage groups. Statistical calculations from the complementation data yielded 17 as the maximum likelihood estimate of the number of pho genes assuming all loci are equally mutable. Most of the phototaxis mutants were found to exhibit bimodal phototaxis and all were found to be impaired in positive thermotaxis supporting convergence of the photosensory and thermosensory pathways. The thermotaxis mutant HPF228 was unaltered in phototaxis suggesting that the mutation in this strain affects a gene product whose site of action is before the convergence of the two pathways. Other phenotypes such as multiple tip formation by aggregates, stumpy fruiting bodies with short or absent stalks and short migration were associated with some pho alleles suggesting multiple biological roles for some gene products important in phototransduction. ---------------------------------------------------------------------- [End CSM-News, volume 2, number 7]