We have developed a system for performing interaction genetics in that

We have developed a system for performing interaction genetics in that uses a cDNA library complementation/multicopy suppression strategy. are required for cytokinesis (Faix et al. 1996). The cortexillins consist of an NH2-terminal spectrin-family actin binding website, a coiled-coil website, and a COOH-terminal website that has DGKD actin filament-bundling activity and a PIP2-binding site. Cortexillin I localizes to the cleavage furrow and the COOH-terminal website is sufficient for cortexillin I’s function in cytokinesis (Weber et al. 1999). This COOH-terminal website is also adequate for actin filament bundling in vitro (Stock et al. 1999). Loss of cortexillin function prospects to a reduction in cortical pressure (Simson et al. 1998). coronin is an actin filament-crosslinking protein that is required to keep up cell shape and loss of it prospects to problems in cytokinesis (deHostas et al. 1991, deHostas et al. 1993). Mutations in the small GTPase gene lead to reduced general cortical pressure and failure in cytokinesis, implicating a signaling cascade that modulates cortical pressure (Larochelle et al. 1997; Gerald et al. 1998). In recent years, many new proteins have been recognized that are involved in or required for cytokinesis. Many of these proteins have been found out in strains, which restricts the ability to map mutations by recombination and greatly inhibits the ability of using genetic recombination to identify epistatic relationships. To dissect further the molecular mechanisms of cytokinesis, we have developed library complementation and high copy suppression methods to carry out connection genetics in cells without the use of the helper pRep plasmid. Table 1 D. discoideum Strains Used in This Paper locus in JH10 cells (referred to as gene Larochelle et al. 1996 p69C5-3G1geneDeLozanne, A., Imiquimod tyrosianse inhibitor personal communicationAx2-214Wild-typeWild-type parent for the cortI-214 strain Faix et al. 1996 cortI-214locus Faix et al. 1996 Ax3:ORF+Wild-type; REP ORF+Ddp2 source replicase ORF integrated in genome (crazy- type normally)Morandini, P., and R. Kay, personal communication; Manstein et al. 1995 HS1000Ax3; ORF+7-3Subclone 7-3 of Ax3:ORF+ cells used as wild-type parental strain for this studyThis paperHS1151cortI11-5.1 NQNO induced deletion in HS1000 Imiquimod tyrosianse inhibitor that removes the locusThis paper Open in a separate windows The myo II null cell collection ((null cell collection (p69C5-3G1) used has a complete deletion of the gene (Table ). The strain is definitely a deletion of the locus generated by homologous recombination in the wild-type Ax2 background (Faix et al. 1996). ORF+7-3Cderived strains were cultivated in Hans’ enriched HL-5 press (1.4 HL-5, 8% FM) plus penicillin (60 U/ml), streptomycin sulfate (60 g/ml), and plus or minus G418 (15 g/ml), depending on whether the cells are transformed with an episomal plasmid. Ax2-214 and JH10 derivatives with episomal plasmids were cultivated in 10 g/ml G418 or 4 g/ml blasticidin, as appropriate. DH1, 24EH6, Imiquimod tyrosianse inhibitor and p69C5-3G1 derivatives with episomal plasmids were cultivated in 7.5 g/ml G418. Cells were propagated at 22C on 10-cm plates. For suspension growth assays, cells were grown in 25-ml tradition quantities in 250-ml Erlenmeyer Imiquimod tyrosianse inhibitor flasks at 180 rpm. Cell densities were determined using a hemacytometer. Mutagenesis and Mutant Screening of D. discoideum Cells For mutagenesis, we altered the protocol explained by Patterson and Spudich 1995. Log phase ORF+7-3 cells were harvested and resuspended in MES starvation buffer (50 mM MES, pH 6.8, 2 mM MgCl2, 0.2 mM CaCl2). 4-nitroquinolone-lawns to select for solitary clones and to assay for developmental phenotypes. For further examination of a cytokinesis phenotype, cell lines were grown in suspension tradition in 25-ml ethnicities in 250-ml Erlenmeyer flasks at 180 rpm. We find that growth under these conditions increases the penetrance of any cytokinesis defect, resulting in multinucleated cells. Cell lines were analyzed for the nuclei/cell percentage distribution, growth rate, smoothness of the growth curve, and saturation denseness. A high nuclei/cell percentage and a lower saturation denseness are indicative of a cytokinesis defect. Plasmids The.