Figure 4

Presence of Sec7 mRNA during D. discoideum development and generation of Sec7 deficient cells. ( A ) Cells were developed on phosphate agar, mRNA was isolated at the indicated time points and RNA used for quantitative Real Time PCR. A 5’ fragment covering the first 500 bases of the cDNA was amplified. Relative values are shown. The mRNA content in growth phase cells (time 0) was arbitrarily set to 1.0 and taken as reference. For internal reference GAPDH amounts were determined. ( B ) Strategy used to generate the Sec7 gene replacement. From top to bottom: position of the nucleotides and the corresponding amino acids; the domain structure of the Sec7 protein; the genomic locus and the replacement vector. The location of the DNA probe used in the Southern blot analysis (3’ probe), the location of the PCR primers used for verification of the gene replacement event and the expected products are shown. ( C ) Southern blot analysis with DNA obtained from two independent clones and AX2 wild type. The DNA was digested with EcoRI and XhoI.