CSM News Electronic Edition Volume 1, number 25 December 10, 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], via Gopher at the same address, or by World Wide Web through WWW.acns.nwu.edu. ========= Abstracts ========= Spatial and temporal expression of the Dictyostelium Ga protein subunit Ga2: Expression of a dominant negative protein inhibits proper prestalk to spore differentiation. Francois Carrel, Suranganie Dharmawardhane, Alexandra M. Clark, Jo Anne Powell-Coffman and Richard A. Firtel Department of Biology, Center for Molecular Genetics, 0634 University of California, San Diego La Jolla, CA 92093-0634 Mol. Biol. Cell, in press ABSTRACT Previous results have shown that the Ga protein subunit Ga2= is required for aggregation in Dictyostelium and is essential for coupling cell-surface cAMP receptors to downstream effectors in vivo during this stage of development. Ga2 expresses at least four distinct transcripts that are differentially regulated during development; two of the transcripts are expressed exclusively in the multicellular stages and their expression is restricted to prestalk cells. We partially dissected the Ga2 promoter and identified a component that is expressed exclusively during the multicellular stages using luciferase gene fusions. When this promoter region is coupled to lacZ, b-gal expression is restricted to the multicellular stages and localized in prestalk cells, with a pattern similar to that of the ecmA prestalk-specific promoter. We show that expression in wild-type cells of the Ga2 mutant protein [Ga2(G206T)] during the early stages of development blocks aggregation and cAMP-mediated activation of adenylyl cyclase and guanylyl cyclase, suggesting it functions as a dominant negatively active Ga subunit. When this mutant Ga protein is expressed from the ecmA prestalk-specfic promoter, abnormal stalk differentiation during culmination is observed. Expression of the mutant Ga2 from the SP60 prespore promoter or wild-type Ga2 from either the ecmA or the SP60 promoter results in no detectable phenotype. The results suggest that Ga2 plays an essential role during the culmination stage in prestalk cells and may mediate cAMP receptor activation of these processes during multicellular development. ============== Technical Tips ============== Karl Saxe writes: Here is a method we have found to be very useful. It is a minor modification of a new genomic miniprep method recently published in Biotechniques. We tend to use 1/2 to all of one of these preps in restriction digests but the cell numbers extracted can be increased easily. The method works equally well for cells growing in flasks or on dishes. Try it! It's quick and relatively painless and works well. Maybe someone else has already played with this method and has modifications or comments of their own. Let everyone know. All comments or reactions are welcome. Microwave Miniprep of Genomic DNA 1) Resuspend 5 x 10E6 cells (either from a dish or shaking flask) in 100ul of lysis solution. Transfer to a PCR tube. 2) With lid open, microwave at full power (sequentially) for 15 seconds, 10 seconds and finally 5 seconds. 3) Add an additional 300ul of lysis solution (or to a total volume of 400ul) and put at 80o C for 10 min. Use PCR program '80C'. 4) Transfer to a new, clear epie (1.7ml) tube and phenol extract with 1:1 ratio. Use pH 8.4 Tris-buffered phenol and vortex for 3 seconds. Centrifuge at 14K for 10 min. 5) Carefully pipet off the aqueous layer into a new epie tube. Add 0.54 volumes (~=175ul) of isopropanol and 10 ul of pH 4.6 3M KOAc. Mix and keep on ice for 10 min. 6) Centrifuge for 6 min. at 14K and carefully remove supernatant by pipeting. (Caution: this pellet is very soft) 7) Wash pellet in 0.5 ml of 70% EtOH and remove supernatant (watch that pellet!). Air dry. Resuspend in 50 ul of DEP H2O for 1 hour on ice. Solutions: Final 1ml 5ml concentration Lysis solution - 1M tris-HCl pH 7.2 50ul 250ul 50mM 0.4M EDTA 125ul 625ul 50mM 20% SDS 150ul 750ul 3% 100% ss-mercaptoethanol 10ul 50ul 1% DEP H2O 665ul 3.325ml Adapted from Goodwin and Lee, BioTechniques Vol. 15, No. 3 (1993) Also just to throw into the mix, here are the methods we have used to do whole cell PCR and releasing cells from the bottom of dishes (questions raised in this forum in the recent past). I claim no special advantage for either of the methods, only that the whole cell PCR method has worked nicely for RACE and genomic DNA amplifications, and the trypsin method provides one more way to harvest cells. Whole Cell Prep : 1) use 1x10E6 cells and place in 1.5 ml eppie tubes 2) spin cells at 1,000 RPM for 10 sec. 3) take media off with a pipet and wash the cell pellet 2x with 750 ul of cold DEPH2O 4) resusp. the cell pellet in 100 ul of PCR buffer (containing no gelatin or BSA) 5) add 2.5 ul of 20% NP-40 (final conc.=0.5%) 6) add 0.1 ul of 50mg/ml Proteinase K (final conc.= 100ug/ml). Yes we generally do this as a dilution. 7) place tube at 56 0 C for 45 min. 8) boil tube for 10 min. (95 deg C) 9) use 10-15 ul of whole cell prep in PCR rxn. PCR Rxn.: 100 ul total volume 10-15 ul cell prep 10 ul Taq or Hot Tub polymerase 10x buffer 2 ul dNTPs (10mM each) *0.5-1.0 ul oligo #1 *0.5-1.0 ul oligo #2 1 ul polymerase enzyme x ml of DEP H2O to bring total volume to 100 ul * Amount of oligos used depends on concentrations. Trypsinising Dictyostelium 1) aspirate media off plates. 2) wash 1X with 1X PBS or HBS 3) aspirate 4) add 3ml 1X trypsin (bought as a 10X stock) in 1X PBS or HBS. 5) incubate 5min @ RT. 6) bang and/or rock the dish to loosen cells, then pipet up and down with a pasteur pipet. 7) add 3ml of axenic media + 2% serum (fbs, fcs, dfbs or others are fine) to stop reaction. 8) count, dilute an go. Cell remaining on dish can be refed and are fine. -------------------------------------------------------------------------- [[END CSM-News, volume 1, number 25]]