CSM News Electronic Edition Volume 2, number 20 June 4, 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 [165.124.233.50], via Gopher at the same address, or by World Wide Web through www.nwu.edu. ========== Abstracts ========== Regulation of Expression of the Cyclic Nucleotide Phosphodiesterase Gene in Phosphodiesterase Inhibitor-negative Mutants of Dictyostelium discoideum Neil R. Adames, M. Barrie Coukell and Lin Wu# Department of Biology, York University, North York, Ontario, M3J 1P3 #Department of Anatomy and Cell Biology, College of Physicians & Surgeons, Columbia University, New York, NY, 10032 Biochem. Cell Biol. - in press Summary During early development of Dictyostelium discoideum, the enzyme cyclic nucleotide phosphodiesterase (PD) is produced at a low rate during the period its specific inhibitor (PDI) is being synthesized. In addition, PD gene expression is derepressed in the aggregation-deficient (Agg-), Pdi- mutant HC35. These observations suggest that the PDI might function to regulate PD gene expression as well as modulate its activity. To explore this idea further, five new Agg-, Pdi- mutants were isolated and analyzed. All of the mutants produced high PD activity and overexpressed PD mRNA; four exhibited elevated levels of the 2,400-nt aggregative transcript, and one overproduced the 1,900-nt vegetative transcript. In contrast, PD transcripts were not elevated in two Agg-, Pdi+ mutants. To determine if PDI production regulates PD expression, HC35 cells were transformed with plasmids carrying the PDI structural gene under the control of either the vegetative or aggregative PD promoter. Neither expression of PDI by the transformants nor addition of partially purified PDI to HC35 cells affected PD transcription. These results suggest that PD overexpression in the Pdi- mutants is not a direct consequence of the inability of these cells to produce inhibitor. ------------------------------------------------------------------------ Serine-113 is the Site of Receptor-Mediated Phosphorylation of the Dictyostelium G protein `-Subunit, G`2 Mei-Yu Chen@, Peter N. Devreotes@ and Robert E. Gundersen# @ Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185 # Department of Biochemistry, Microbiology and Molecular Biology University of Maine, Orono, Maine 04469-5735 J. Biol. Chem., in press. SUMMARY The G protein `-subunit, G`2, is essential to the developmental program of Dictyostelium. G`2 is transiently phosphorylated on a serine residue(s) following stimulation with extracellular cAMP (Gundersen, R.E. and Devreotes, P.N. (1990) Science 248, 591-593). To aid in defining the function of `-subunit phosphorylation, we identified the site of G`2 phosphorylation. Comparison of the isoelectric points (pI) of the phosphorylated and nonphosphorylated forms indicated that a single mole of phosphate is added to G`2. Cleavage at tryptophan residues and immunoprecipitation with a specific peptide antibody localized the phosphorylated serine in the N-terminal 119 residues. Analysis of a series of G`1 and G`2 `-subunit chimeras further confined the site between amino acids 33 and 215. Site-directed mutagenesis of serines between amino acids 33 to 119 produced two mutants that were not phosphorylated, S45A and S113A. S113 was identified as the site by sequential Edman degradation of 32P-radiolabeled G`2 digested with endoproteinase Glu-C. We have expressed the G`2 mutants S113A, S113I, S113T, and S113D in a G`2 null cell line to examine the function of phosphorylation. -------------------------------------------------------------- REMI-RFLP Mapping in the Dictyostelium Genome Adam Kuspa*, and William F. Loomis Center for Molecular Genetics, Department of Biology, University of California, San Diego, La Jolla, CA 92093-0322 *Present address: Department of Biochemistry, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 Genetics, in press. ABSTRACT A set of 147 Dictyostelium discoideum strains was constructed by random integration of a vector containing rare restriction sites. The strains were generated by transformation using restriction enzyme mediated integration (REMI) which results in the integration of linear DNA fragments into randomly distributed genomic restriction sites. Restriction fragment length polymorphism (RFLP) was generated in a single genomic site in each strain. These REMI-RFLP strains were used to confirm gene linkages previously supported by two other physical mapping techniques: Yeast Artificial Chromosome (YAC) contig construction, and megabase-scale restriction mapping. New linkages were uncovered when two or more hybridization probes identified the same RFLP fragments. Probes for 100 genes have marked 53% of the RFLPs, representing greater than 22 Mb of the 40 Mb Dictyostelium genome. Alignment of these and other large fragments along each chromosome should lead to a complete physical map of the Dictyostelium genome. -------------------------------------------------------------------------