Fig. 1. MidA disruption and expression. (A) The sequence of Dictyostelium MidA protein was aligned with the human protein PRO1853 (GenBank accession number: NP_653337) and the most similar prokaryote protein from the alpha-proteobacteria Magnetospirillum magnetotacticum (GenBank accession number: ZP_00207869) using the ClustalW program. Black background corresponds to identical residues in three species, dark gray to identical residues in two species and light gray to similar residues. (B) The coding region of midA gene is depicted as two open boxes. The line between the open boxes represents an intron. KO1 and KO2 are two mutant strains generated by disruption of the midA coding region by homologous recombination at the indicated locations. Horizontal arrows show the oligonucleotides (1-4) used for checking the disruption of midA genomic locus in these strains. (C) Disruption of the midA gene was assessed by PCR. DNA from wild-type, KO1 (right panel) and KO2 (left panel) strains were subjected to PCR using the indicated oligonucleotides. A pair of oligonucleotides from an unrelated locus was used as internal control of the PCR reaction. The expected bands were absent in the KO strains due to the insertion of the BS-cassette. The upper bands in the KO samples (labeled as BS) corresponded to the inserted cassette. (D) RNA isolated at different times of development was hybridized with a radioactive probe derived from the coding sequence of the midA gene (upper panel). Lower panel shows the absence of midA mRNA in RNA samples from the KO strains isolated from vegetative stage.